Methods of isolating amyloid-inhibiting compounds and use of compounds isolated from Uncaria tomentosa and related plants

ABSTRACT

Assay-guided affinity fractionation and reverse phase high pressure liquid chromatography (HPLC) methodology to isolate, test and characterize the most active water-soluble ingredients within Cat&#39;s Claw, or  Uncaria tomentos.  These components appear to account for the majority of the amyloid or Aβ fibrillogenesis inhibitory activity. Individual fractions and/or compounds as isolated by HPLC are tested in relevant in vitro and/or animal models, and found to consistently demonstrate inhibition of amyloid or Aβ fibrillogenesis. Related extraction methods are disclosed.

[0001] This application claims priority to US provisional applications60/245958 filed Nov. 3, 2000, and 60/271777 filed Feb. 27, 2001.

TECHNICAL FIELD

[0002] The invention relates to the method of isolation and use ofamyloid-inhibiting compounds derived from Uncaria tomentosa and relatedplants for the therapeutic intervention of Alzheimer's disease, type IIdiabetes, Parkinson's disease and other disorders involving amyloidaccumulation; more particularly, it relates to methods of isolatingamyloid-inhibiting compounds from Uncaria tomentosa and related plants,and to the use of those compounds.

BACKGROUND OF THE INVENTION

[0003] Alzheimer's disease is characterized by the accumulation of a39-43 amino acid peptide termed the beta-amyloid protein or Aβ, in afibrillar form, existing as extracellular amyloid plaques and as amyloidwithin the walls of cerebral blood vessels. Fibrillar Aβ amyloiddeposition in Alzheimer's disease is believed to be detrimental to thepatient and eventually leads to toxicity and neuronal cell death,characteristic hallmarks of Alzheimer's disease. Accumulating evidenceimplicates amyloid as a major causative factor of Alzheimer's diseasepathogenesis.

[0004] A variety of other human diseases also demonstrate amyloiddeposition and usually involve systemic organs (i.e. organs or tissueslying outside the central nervous system), with the amyloid accumulationleading to organ dysfunction or failure. In Alzheimer's disease and“systemic” amyloid diseases, there is currently no cure or effectivetreatment, and the patient usually dies within 3 to 10 years fromdisease onset.

[0005] The amyloid diseases include, but are not limited to, the amyloidassociated with Alzheimer's disease, Down's syndrome and hereditarycerebral hemorrhage with amyloidosis of the Dutch type (wherein thespecific amyloid is referred to as beta-amyloid protein or Aβ), theamyloid associated with chronic inflammation, various forms ofmalignancy and Familial Mediterranean Fever (wherein the specificamyloid is referred to as AA amyloid or inflammation-associatedamyloidosis), the amyloid associated with multiple myeloma and otherB-cell dyscrasias (wherein the specific amyloid is referred to as ALamyloid), the amyloid associated with type II diabetes (wherein thespecific amyloid protein is referred to as amylin or islet amyloidpolypeptide), the amyloid associated with the prion diseases includingCreutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru andanimal scrapie (wherein the specific amyloid is referred to as PrPamyloid), the amyloid associated with long-term hemodialysis and carpaltunnel syndrome (wherein the specific amyloid is referred to asbeta2-microglobulin amyloid), the amyloid associated with senile cardiacamyloid and Familial Amyloidotic Polyneuropathy (wherein the specificamyloid is referred to as transthyretin or prealbumin), and the amyloidassociated with endocrine tumors such as medullary carcinoma of thethyroid (wherein the specific amyloid is referred to as variants ofprocalcitonin).

[0006] Discovery and identification of new compounds or agents aspotential therapeutic agents to arrest amyloid formation, deposition,accumulation and/or persistence that occurs in Alzheimer's disease,Parkinson's disease and other amyloidoses are desperately sought.

DISCLOSURE OF THE INVENTION

[0007] Methods of isolation for the identification and purification ofthe potent amyloid inhibitory ingredients within Uncaria tomentosa andrelated plants are disclosed. Use of such extracts from the inner barkand root parts of Uncaria tomentosa and related plant materials areanticipated to benefit human patients with Alzheimer's disease, type IIdiabetes, Parkinson's disease and other amyloidoses, due to thepreviously unknown ability of these compounds to inhibit amyloid fibrilformation, and cause disruption/ dissolution of pre-formed amyloidfibrils.

[0008] The present invention pertains to the surprising discovery thatspecific extraction methods (and compounds derived from such extractionmethods) when applied to the inner bark and root parts of Uncariatomentosa, otherwise known as Uña de Gato (or Cat's claw), leads to thepurification of a group of compounds (the group referred to herein asPTI-777), and their individual components (such as “compound H”) whichact as impressive inhibitors of Alzheimer's disease beta-amyloid protein(Aβ) formation and growth.

[0009] Previously our studies led to the identification of a naturalsubstance derived from the Amazon rain forest woody vine, Uncariatomentosa, and referred to as PTI-00703. See for instance U.S. patentapplications Ser. Nos. 09/079,829, 09/198,824, and 09/208,278, whichdescribe the initial discovery of derivatives of Uncaria tomentosa andrelated plant material extracts as inhibitors of amyloidosis ofAlzheimer's disease, type II diabetes and other amyloid disorders.

[0010] In the present application, we used assay-guided affinityfractionation and reverse phase high pressure liquid chromatography(HPLC) methodology to isolate, test and characterize the most activewater-soluble ingredients within PTI-00703 (collectively referred to asPTI-777) that appear to account for the majority of the ADfibrillogenesis inhibitory activity. PTI-777 and its individualfractions and/or compounds as isolated by HPLC were tested in relevantin vitro and/or animal models, and found to consistently demonstrateinhibition of Aβ fibrillogenesis. The present invention describesextraction methods for the isolation of PTI-777 and its individualfractions and/or components.

[0011] Further purification and in vitro testing of each of the PTI-777compounds, as well as initial structural characterization studiessuggest that the Aβ inhibitor compounds derived from Uncaria tomentosaare small molecules (˜200-500 molecular weight) that belong to thegeneral class of aromatic polyphenolic compounds. Two such compounds,chlorogenic acid (C₁₆H₁₈O₉; FW 354.31) and epicatechin (C₁₅H₁₄O₆; FW290.27) were purified and identified by analytical techniques. Inaddition, data indicates that “compound H”, the major compound within“fraction H” isolated from PTI-777 is a most potent inhibitor of Aβamyloid fibrillogenesis.

[0012] In addition, PTI-777 has the ability to enter the brain asdemonstrated by radiolabeling experiments, indicating that it has thepotential to be very useful as a therapeutic agent for Alzheimer'sdisease, Parkinson's disease, and other central nervous system disordersinvolving deposition and accumulation of fibrillar proteins.

[0013] A primary object of the present invention is to establish newmethods for the treatment of the amyloid diseases. In addition, thealpha-synuclein protein which forms fibrils, and is also Congo red andThioflavin S positive, is found as part of Lewy bodies in the brains ofpatients with Parkinson's disease (Lewy in Handbuch der Neurologie, M.Lewandowski, ed., Springer, Berline pp.920-933, 1912; Pollanen et al, J.Neuropath. Exp. Neurol. 52:183-191, 1993; Spillantini et al, Proc. Natl.Acad. Sci. USA 95:6469-6473, 1998; Arai et al, Neurosc. Lett. 259:83-86,1999). For purposes of this disclosure, Parkinson's disease, due to thefact that fibrils develop in the brains of patients with this disease(which are Congo red and Thioflavin S positive, and which also containpredominant beta-pleated sheet secondary structure), are regarded as adisease that also displays the characteristics of an amyloid-likedisease.

[0014] Yet another object of the present invention is to use fraction“H” contained within Uncaria tomentosa and related plant materials forthe treatment of amyloid formation, deposition, accumulation and/orpersistence in Alzheimer's disease, type II diabetes, other amyloidosesand Parkinson's disease.

[0015] Yet another object of the present invention is to provide methodsto isolate the active water-soluble amyloid inhibitory ingredientspresent within Uncaria tomentosa and related plant materials for use aspotent agents which inhibit amyloid formation, amyloid deposition,amyloid accumulation, amyloid persistence, amyloid protein-amyloidprotein interactions, and/or cause a dissolution/disruption ofpre-formed or pre-deposited amyloid fibrils in Alzheimer's disease, typeII diabetes, other amyloidoses and Parkinson's disease.

[0016] Yet another object of the present invention is to providecompositions and methods involving administering to a subject atherapeutic dose of an Uncaria tomentosa and related plant materialextract which inhibits amyloid deposition. Accordingly, the compositionsand methods of the invention are useful for inhibiting amyloidosis indisorders in which amyloid deposition occurs. The compounds of theinvention can be used therapeutically to treat amyloidosis or can beused prophylactically in a subject susceptible to amyloidosis. Themethods of the invention are based, at least in part, in directlyinhibiting amyloid fibril formation, inhibiting amyloid fibril growth,and/or causing dissolution/disruption of preformed amyloid fibrils.

[0017] Yet another object of the present invention is to providepharmaceutical compositions for treating amyloidosis. The pharmaceuticalcompositions include a therapeutic compound of the invention in anamount effective to inhibit amyloid deposition and a pharmaceuticallyacceptable vehicle.

[0018] Yet another object of the present invention is the use of any andall synthetic compounds made similar to an Uncaria tomentosa and relatedplant material extract for use as potent agents which inhibit amyloidformation, amyloid deposition, amyloid accumulation, amyloidpersistence, amyloid protein-amyloid protein interactions, and/or causea dissolution/ disruption of pre-formed or pre-deposited amyloid fibrilsin Alzheimer's disease, type II diabetes, other amyloidoses andParkinson's disease.

[0019] In a particular aspect of the invention there is a method ofisolation to purify and identify the water-soluble amyloid inhibitoryingredients from Uncaria tomentosa and/or extracts thereof. In one suchmethod, an extract prepared from commercially obtained pills, tablets,caplets, soft and hard gelatin capsules, lozenges, sachets, cachets,vegicaps, liquid drops, elixers, suspensions, emulsions, solutions,syrups, tea bags, aerosols (as a solid or in a liquid medium),suppositories, sterile injectable solutions, sterile packaged powders,bark bundles and/or bark powder, using the methods described in thepresent invention.

[0020] Another object of the present invention is to use the methods ofextraction as described herein to provide an extract from Uncariatomentosa and related plant materials for promoting mental alertness andfor inhibiting the formation of brain amyloid deposits in a subject.

[0021] Yet another object of the present invention is to use the extractfrom Uncaria tomentosa and related plant materials for mental acuity; topromote mental alertness; to provide nutritional support for age orrelated cognitive or memory decline; to promote cognitive well being; tosupport brain function; to improve cognitive ability, mental performanceor memory; to promote concentration and mental sharpness; to improvemental vitality; to promote greater mental clarity and alertness; toimprove short term memory, for age associated cognitive or memorydecline; to support normal brain function; to enhance learning ormemory; to improve concentration; to enhance mental performance; toreduce mental decline; to reduce likelihood of age related braindisorders; to maintain good brain health; to reduce, eliminate, prevent,inhibit or disrupt/dissolve amyloid fibril or protein deposits, brainassociated amyloid fibril deposits or brain associated amyloid proteindeposits, amyloid fibril formation and growth or age associated amyloidfibril formation and growth, brain associated amyloid fibril formationand growth; to support healthy pancreatic function; to promotepancreatic function by helping to promote normal insulin function; toreduce, eliminate, prevent, inhibit or disrupt/dissolve amyloid fibrilor protein deposits, and pancreas associated amyloid fibril formationand growth.

[0022] In particular the disclosure is directed to novel applications ofassay guided fractionation leading to novel compounds and novel methodsof use of those novel compounds, such as:

[0023] A method for isolating compounds that possess amyloid inhibitoryactivity from plant matter of the genus Uncaria having the followingsteps:

[0024] a) preparing a polar solvent extract (preferably a methanolextract) of Uncarica plant matter, where the polar solvent extraction isextraction with water, extraction with a polar alcohol or a watersolution of a polar alcohol, extraction with a water solution ofacetonitrile, or extraction with a water solution of another polarorganic solvent such as triethanolamine, acetone, or the like, andrunning the extract through a first column that has a hydroxy groupcontaining resin, a resin having hydrophobic characteristics but withoutany hydroxy groups, or a mixture of both;

[0025] b) eluting the first column with distilled water, followed byeluting with not more than 2-4 column bed volume washings with a dilutepolar alcohol (such as methanol)/water solution having an alcohol/waterratio not greater than about 50/50, depending on which alcohol is used,and discarding any eluate, the object being to wash non-active materialand fractions away, without appreciably eluting any active fractions(during particularization of any separation protocol—choice of solventand concentration, volume of washings, flow rates and the like,appropriate analytical testing of putative discardable eluates isdesirable, as will be appreciated by those skilled in the art, and suchpersons will know what tests to perform, such as for instance standardThioflavin T testing to detect amyloid inhibiting substances);

[0026] c) eluting the first column with one or more column bed volumewashings of a polar alcohol/water solution having an alcohol/water ratiosomewhere at, or between, 50/50 and substantially pure alcohol, andcollecting and drying the eluted volumes to a dried material. Thesevolumes and their dried material contain the active amyloid inhibitingingredients referred to in this disclosure as PTI-777.

[0027] It will be appreciated that in the drying step above, alternateconventional drying procedures may be substituted by those skilled inthe art without departing from the scope of coverage, and in some cases,the drying step may be omitted

[0028] In the method of above, the column that comprises hydroxycontaining resin, resin having hydrophobic characteristics but withoutany hydroxy, or a mixture of both, may advantageously be a column suchas a C2 column, C4 column, C18 column, or the like, or Tris-acrylatecolumn, LH-20 column, Affi-prep 10 gel column, or the like. Also thepolar alcohol/water solution preferably has an alcohol/water ratio of75/25 or higher, and more preferably is pure or nearly pure alcohol, andpreferably methanol.

[0029] The plant matter of the genus Uncaria is preferably taken fromone or more of the various Uncaria species such as tomentosa, attenuata,elliptica, guianensis, pteropoda, bernaysli, ferra DC, kawakamii,rhyncophylla, calophylla, gambir, and orientalis, and more preferably,Uncaria tomentosa. The Uncaria tomentosa plant matter is preferablytaken from the inner bark and/or the root.

[0030] Optionally the isolation method set forth above is extended withthe further steps:

[0031] d) applying an aqueous solution of the dried material from step(c) to a second column comprising a hydrophobic resin, the second columnhaving been preparatorily equilibrated in a solvent comprising about 95%water/5% acetonitrile, referred to herein as solvent A, and then elutingthe second column with more solvent A and discarding the eluate.

[0032] e) eluting the second column with a mixture of solvent Acontaining about 10-15%, and preferably about 12.5%, of a solventcomprising about 95% acetonitrile/5% water, referred to herein assolvent B, and collecting and drying the eluted volumes to a driedmaterial.

[0033] “About” as applied to solvent percentage compositions andgenerally to other percentages expressed in this disclosure generallyrefers to +/− about 2% points; thus ‘about 95% water/5% acetonitrile’,for example, can lie anywhere at or between 97% water/3% acetonitrile to93% water/7% acetonitrile. In other instances, the words ‘about’ or‘substantially’ are understood to mean a figure or amount somewhereclose to the stated figure, varying from the stated figure or amount byas much as +/−5%-20% of the stated figure or amount.

[0034] Optimally, TFA (typically about 0.1%) is added to the solventsindicated for acid stability and added efficacy in resin column work, aswill be appreciated by those skilled in the art.

[0035] The isolation method above may be yet further advantageouslyenhanced by having a hydrophobic resin in the second column, andselecting a column from one of the many so called ‘carbon columns’, orcarbon/hydrophobic columns, each preferably containing no hydroxygroups, such as for instance a C18 SPE, Varian Chroma..Zone™, or otherHPLC columns, or the like.

[0036] The isolation method above may be yet further advantageouslyextended by having the following additional steps:

[0037] f) making one or more injections of a solution of the driedmaterial of step (c) or the dried material of step (e) in a solvent suchas water, water/dilute alcohol or a solution of solvent A comprising nomore than 10% solvent B, into an HPLC instrument with a diode arrayuv/vis detector and graphic display and a reverse-phase column;

[0038] g) eluting the material through the HPLC column using a solventgradient profile as follows: 10% solvent B for about the first 20minutes from start of elution, 10 to 100% solvent B gradient for aboutminutes 20 to 30 from start of elution, and 100 to 10% solvent Bgradient for about minutes 30 to 32 from start of elution, whileobserving the uv/vis detector graphic display during the elutiongradient over time, and separating fractions of the eluate at elutiontimes corresponding to times associated with the graphic display peaks.

[0039] Suitable reverse phase columns will occur to, and be well knownby, those skilled in the art, and with minor adjustments to the protocoldescribed above, may be interchanged for any columns set forth here. Asdiscussed above, one of the many so called ‘carbon columns’, orcarbon/hydrophobic columns, each preferably containing no hydroxygroups, such as for instance a C18 SPE, Varian Chroma. .Zone™, or otherHPLC columns, or the like, may be employed.

[0040] It should be noted that the preferred diode array detector may beadvantageously substituted with alternate detectors such as a RI(refractive index) detector, a total ion detector, or the like, in orderto monitor and record intensity peaks over time that correspond toelution fractions, as does the uv/vis detector preferred.

[0041] In a particular embodiment, the reverse-phase column hasdimensions of about 2.2 cm×25 cm and contains about 95 ml of C18 reversephase resin. The solution of the dried material is advantageously asolution of about 50 mg of the dried material of step (c) in about 1-2ml of solvent A, and the step of injecting the solution of driedmaterial into the HPLC may be repeated as required to load the column.An HPLC column solution gradient flow rate is preferably set to about 5mls per minute, and the solvent gradient profile is preferably 10%solvent B for 0 to 20 minutes, followed by 10 to 100% solvent B gradientfor minutes 20 to 30, and 100% to 10% solvent B gradient from minutes 30to 31; such that fractions F though N of the eluate are collected at thefollowing times: fraction G (13-14 minutes), fraction F (15-16 minutes),fraction H (17-20 minutes), fraction I (21 minutes), fraction J (22-23minutes), fraction K1 (24 minutes), fraction K2 (25 minutes), fraction L(26-27 minutes), fraction M (27-28 minutes), and fraction N (28-29minutes).

[0042] In another embodiment, the reverse-phase column has dimensions of1.0 cm×25.0 cm and contains about 20 ml of C18 reverse phase resin. Thesolution of the dried material of step (c) is a solution of about 50 μgof the dried material in 50-100 μl of solvent A, wherein the step ofinjecting the solution into the HPLC is repeated multiple times, whereina HPLC column solution gradient flow rate is set to about 1.5 mls perminute, and further wherein the solvent gradient profile is 10% solventB for 0 to 20 minutes, followed by 10 to 100% solvent B gradient forminutes 20 to 30, and 100% to 10% solvent B gradient from minutes 30 to31; such that fractions F though O of the eluate are collected at thefollowing times: fraction G (12-13 minutes), fraction F (13-14 minutes),fraction H (15 minutes), fraction I (16 minutes), fraction J (18-19minutes), fraction K1 (20 minutes), fraction K2 (21 minutes), fraction L(21-23 minutes), fraction M (23 minutes), fraction N (24 minutes), andfraction O (26-27 minutes).

[0043] Steps (f) and (g) of the isolation method set forth above mayalternatively proceed as follows:

[0044] f) injecting a solution of 1 gram of the dried material of step(c) in 5-10 ml of solvent A into an HPLC instrument having a Varianmodel 320 uv/vis detector set at 230 nm with a graphic display, the HPLCfurther comprising a 4.14 cm×25 cm Varian Dynamax column furthercomprising 380 ml of C-18 reverse phase resin, the column fitted to aVarian Prostar 215 solvent delivery system, or the like.

[0045] g) eluting the HPLC column at a solution gradient flow rate ofabout 50 ml/mlnute, and further wherein the solvent gradient profile iswith a solvent C/solvent D gradient (referred to in the art for HPLCsolvent gradients as “A/B”, but as C/D here to avoid confusions withother A/B gradients referred to herein as standards of protocolelsewhere in this disclosure) as follows: 0-4 minutes, 25% D; 4-11minutes, 25-30% D gradient; 11-14 minutes, 30-90% D gradient; 14-17minutes, 90% D; and 17-19 minutes, 90-25% D gradient, where C is waterand D is methanol, such that fractions F through O of the eluate areseparated at elution times corresponding to times associated with thegraphic display peaks.

[0046] Those skilled in the art will appreciate, and readilyaccommodate, without undue experimentation, that adjusting flow ratesand gradients for substitution of various A/B gradient setups, such assubstituting water/methanol for water/acetonitrile, will be necessaryand appropriate, because for instance methanol is more polar thanacetonitrile, and thus more methanol (25%) is needed compared toacetonitrile (10%) in the discussions herein. Even so, specificpercentages, times and flow rates will readily be selectable for variouschoices of solvents, all in accordance with the teachings disclosedherein.

[0047] Alternatively the preparation in step (a) of the extract ofUncaria may proceed as follows:

[0048] 1) adding 4000 ml of methanol to 1 kg of Uncaria tomentosa andmixing

[0049] 2) centrifuging the mixture at ×2,500 g using a centrifuge for 30minutes and collecting the supernatant;

[0050] 3) extracting the insoluble material about 3 more times as stepsa and b above;

[0051] 4) combining the supernatants and evaporating to a dried extract,or to at least about 500 ml volume, using a rotary evaporator at 50° C.;

[0052] 5) washing the dried extract, or the 500 ml volume, 4 times with300 ml of petroleum ether, and discarding the ether layer;

[0053] 6) further evaporating any remaining methanol to dryness using arotary evaporator at 50° C.;

[0054] 7) extracting the dried extract 5 times with 150 ml of distilledwater, followed by centrifugation at 2,500×g for 30 minutes each time,and

[0055] 8) combining the supernatants and then lyophilizing using afreeze-dryer. Further preparation of the extract of Uncaria from theresulting lyophilized extract can use the following additional steps:

[0056] 9) dissolving the resulting lyophilized extract into about 500 mlof distilled water, and applying 50-100 ml portions to a 400 ml LH-20column equilibrated with distilled water.

[0057] 10) eluting the LH-20 column with 1,100 ml of distilled water (˜3column volumes) and discarding the amber/yellow, non-active fractions;

[0058] 11) eluting the LH-20 column with 1,100 ml of 100% methanol (˜3column volumes) and collecting a set of active fractions and evaporatingto dryness using a rotary evaporator at 50° C.

[0059] Alternatively the aqueous solution of a dried material from step(c) may be further prepared by the following steps:

[0060] 1) dissolving the dried material in water at 80 mg/ml andapplying 5 ml at a time to a disposable C18 SPE column (10 gram)equilibrated in a first solvent comprising about 95% water/5%acetonitrile/0.1% TFA;

[0061] 2) washing with 3 column bed volumes of the first solvent anddiscarding the eluate.

[0062] 3) eluting with 3 column bed volumes of the first solvent furthercomprising about 12.5% of a second solvent comprising about 95%acetonitrile/5% water/0.1% TFA, and

[0063] 4) lyophilizing the corresponding fractions using a freeze-dryer.

[0064] It will be appreciated that the various drying and volumereducing methods disclosed are well known to those skilled in the art,and effective substitutes are also well known. Other drying methods,where at least one object is to avoid oxidation of the extractedmaterial, such as nitrogen atmosphere, or vacuum drying will occur tothose skilled in the art without departing from the scope of inventionset forth herein.

[0065] Alternately the aqueous solution of a dried material from step(c) is further prepared by the following steps:

[0066] 1) dissolving the lyophilized fractions at 5 grams in 20 ml waterand applying 20 ml at a time to a Varian Chroma..Zone ™apparatus

[0067] 2) washing with 3 column bed volumes of a first solventcomprising about 95% water/5% acetonitrile/ 0.1% TFA and discarding theeluate;

[0068] 3) eluting with 3 column bed volumes of the first solvent furthercomprising about 12.5% of a second solvent comprising about 95%acetonitrile/5% water/0.1% TFA, and

[0069] 4) collecting and drying the next 3 column bed volumes of eluate.

[0070] Another, more particular, method for isolating water-solublecomponents from Uncaria tomentosa that possess amyloid inhibitoryactivity has the following steps:

[0071] a) adding 4000 ml of methanol to 1 kg of Uncaria tomentosa andmixing

[0072] b) centrifuging the mixture at ×2,500 g using a centrifuge for 30minutes and collecting the supernatant, where it is understood thatmeans for separating suspended matter from the liquid, such as overnightsedimentation by gravity or filtration may be substituted by thoseskilled in the art to separate suspended solids from solution,

[0073] c) extracting the insoluble material about 3 more times as stepsa and b above;

[0074] d) combining the supernatants and evaporating to dryness (oruntil about 500 ml volume is reached) using a rotary evaporator at 50°C.,

[0075] e) taking the powdered extract (or about 500ml volume), washing 4times with 300 ml of petroleum ether, or other non-polar organicsolvent, and discarding the ether (non-polar) layer,

[0076] f) evaporating the methanol to dryness using a rotary evaporatorat 50° C.;

[0077] g) extracting the solid material 5 times with 150 ml of distilledwater, followed by centrifugation at 2,500×g for 30 minutes each time;

[0078] h) combining the supernatants and then lyophilizing using afreeze-dryer;

[0079] i) dissolving the resulting lyophilized extract into about 500 mlof distilled water, and applying 50-100 ml portions to a 400 ml LH-20column equilibrated with distilled water.

[0080] j) eluting the LH-20 column with 1,100 ml of distilled water (˜3column volumes) and discarding the amber/yellow, non-active fractions;

[0081] k) eluting the LH-20 column with 1,100 ml of 100% methanol (˜3column volumes) and collecting a set of active fractions and evaporatingto dryness using a rotary evaporator at 50° C.;

[0082] l) dissolving the fractions of step k in water (80 mg/ml) andapplying 5 ml at a time to a 10 gm disposable C18 SPE columnequilibrated in solvent A (solvent A is 95% water/5% acetonitrile/0.1%TFA);

[0083] m) washing the column with 3 volumes of solvent A and discardingthe eluate;

[0084] n) eluting the column with 3 volumes of solvent A containing12.5% solvent B (solvent B is 95% acetonitrile/5% water/0.1% TFA) andlyophilizing the eluate;

[0085] o) taking 50 mg of the lyophilized eluate of step n and injectingmultiple times into a Hewlett-Packard 1100 Series HPLC instrument withdiode array detector, fitted with a 2.2 cm×25 cm Vydac 218TP1022 C18reverse-phase column maintained at 25° C. and at a flow rate of 5ml/min;

[0086] p) eluting the sample with the following solvent profile, 10% Bfor 0 to 20 minutes, 10-100% B gradient for minutes 20 to 30, and100-10% B gradient for minutes 30-31, where B is 95% acetonitrile/5%water/0.1% TFA;

[0087] q) and separating and collecting the fractions into 11 majorcomponents defined as fraction G (13-14 minutes), fraction F (15-16minutes), fraction H (17-20 minutes), fraction I (21 minutes), fractionJ (22-23 minutes), fraction K1 (24 minutes), fraction K2 (25 minutes),fraction L (26-27 minutes), fraction M (27-28 minutes), and fraction N(28-29 minutes).

[0088] A novel composition further referred to herein as PTI-777 maythus be isolated according to any of the isolation processes set forthabove. And other compositions further referred to herein as PTI-777fractions, such as PTI-777 fraction G, PTI-777 fraction F, PTI-777fraction H, PTI-777 fraction I, PTI-777 fraction J, PTI-777 fraction K₁,PTI-777 fraction K₂, PTI-777 fraction L, PTI-777 fraction M, PTI-777fraction N, and PTI-777 fraction O, may also be isolated according toany of the processes set forth above that employ HPLC fractionation.

[0089] A further novel compound H may be isolated by a method havingsteps (a) through (c) as set forth above, and further having the steps:

[0090] d) applying an aqueous solution of the dried material from step(c) to a second column, LH-20 or the like, eluting the material from thecolumn with successive column volumes of water/methanol mixturescontaining 0.1% TFA, beginning with 25% methanol and increasing to 100%menthol in 25% increments, and collecting and combining the fractions;

[0091] e) separating, combining and drying a fraction to a driedmaterial, referred to hereafter as compound H, by analytical HPLC, thefraction containing a peak occurring between 7-8 minutes from start ofelution on a Dynamax 5 μ C-18 column having dimensions of about 4.6mm×25 cm, using an elution gradient of water for solvent A and methanolfor solvent B, A and B each containing about 0.1% TFA, with detection at280 nm, the gradient conditions being 0 to 9 min fro 25% to 36% Bgradient, 3 to 10 min for 36 to 100% B gradient, 10 to 12 min for 100% Band 12 to 13 min for 100 to 25% B gradient, all at a flow rate of about20 ml/min;

[0092] f) making one or more injections of a solution of the driedmaterial of step (e) above in a solvent comprising water/methanol 80/20containing about 0.1% TFA and applied at about 150 mg/run to apreparative HPLC Dynamax 5 μ C-18 column with dimensions of about 21.4mm×25 cm, using substantially the same elution gradient as used in step(e) above, with detection at 280 and 300 nm, the gradient conditionsbeing 0 to 3 min for 20% to 25% B gradient, 3 to 9 min for 25 to 45% Bgradient, 9 to 10 min for 45 to 100% B gradient, 10 to 12 min for 100% Band 12 to 13 min for 100 to 25 % B gradient, all at a flow rate of about20 ml/min, the compound H fraction eluting between 7-8 minutes fromstart of elution, and;

[0093] g) repeating steps (e) and (f) above until the peak as seen onanalytical HPLC in step (e) is relatively pure, thus ending, whenappropriately dried, with substantially pure compound H.

[0094] Also disclosed is a method of treatment, prevention or managementof an amyloidosis, or a disease related to alpha-synuclein, in amammalian subject susceptible to, or afflicted by, the amyloidosis oralpha-synuclein disease. The method includes the step of administeringto the subject a therapeutic amount of the composition produced inaccordance with any of the methods set forth above, such as, inparticular, PTI-777 and/or compound H., or any of fraction G, fractionF, fraction H, fraction I, fraction J, fraction K₁, fraction K₂,fraction L, fraction M, fraction N or fraction O.

[0095] This method may be efficaciously applied to any amyloidosis whichhas an associated amyloid, such as amyloidoses associated withAlzheimer's disease, Down's syndrome, hereditary cerebral hemorrhagewith amyloidosis of the Dutch type, the amyloidosis associated with typeII diabetes, the amyloidosis associated with chronic inflammation,various forms of malignancy and Familial Mediterranean Fever, theamyloidosis associated with multiple myeloma and other B-celldyscrasias, the amyloidosis associated with the prion diseases includingCreutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru andanimal scrapie, the amyloidosis associated with long-term hemodialysisand carpal tunnel syndrome, the amyloidosis associated with endocrinetumors such as medullary carcinoma of the thyroid, and thealpha-synuclein associated diseases including Parkinson's disease andLewy body disease, and in particular, Alzheimer's disease.

[0096] In this method the associated amyloid may be either beta-amyloidprotein or Aβ, AA amyloid or inflammation-associated amyloid, ALamyloid, amylin or islet amyloid polypeptide, PrP amyloid,beta₂-microglobulin amyloid, transthyretin or prealbumin, or variants ofprocalcitonin.

[0097] Another method for the treatment, inhibition, prevention ormanagement of amyloid fibril or alpha-synuclein fibril formation,deposition, accumulation, aggregation and/or persistence in a mammaliansubject is disclosed, and the method includes the step of administeringto the subject a therapeutic amount of any of the compositions isolatedby any of the methods disclosed herein. Contemplated routes ofadministration of the method of treatment include oral administration,parenteral injection, intraperitoneal injection, intravenous injection,subcutaneous injection, or aerosol spray administration.

[0098] A novel pharmaceutical agent is disclosed that is comprised of atherapeutically effective amount of a material made according to any ofthe disclosed isolation processes, with the therapeuti c amount of thematerial selected for efficacy in treating an amyloid disease in apatient.

[0099] Another pharmaceutical agent is disclosed that is comprised of atherapeutically effective am ount of a chlorogenic acid and/orepicatechin, the compound and the therapeutic amount of the compoundselected for efficacy in treating an amylold disease in a patient.

[0100] In either or both of the pharmaceutical agents disclosed above,the therapeutically effective amount of a material is a dosage in therange of from about 10 to 1,000 mg/kg of body weight of the patient, andmore particularly from about 10 to 100 mg/kg of body weight of theparent. The pharmacological agent may also contain a pharmaceu tically acceptable carrier, diluent, or excipient. A therapeutically effectiveamount of the material is defined as an amount that has an amyloidinhibitory activity or efficacy greater than 50%, as compared toplacebo, or no material at all.

[0101] It is yet another object of the invention to meet any or all ofthe needs summarized above.

[0102] These and other features and advantages of the present inventi onwill becom e more fully apparent when the following detailed descriptionof the invention is read in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0103]FIG. 1 is a Black and White Graph of a Thioflavin T FluorometryAssay used to Identify Water-Soluble Fractions of Uncaria tomentosa thatPossess Beta-Amyloid Protein (Aβ) Fibril Disruption Abilities.

[0104]FIG. 2 is a Preparative HPLC Profile of PTI-777 (Batch II) whichDemonstrates the Major Water-Soluble Amyloid Inhibitory Comp onentsIsolated form Uncaria tomentosa.

[0105]FIG. 3 is a Preparative HPLC Profile of PTI-777 (Batch I) that Demonstrates the Maj or W ater-Soluble Amyloid Inhibitory ComponentsIsolated from Uncaria tomentosa.

[0106]FIG. 4 is a Black and White Graph of a Thioflavin T FluorometryAssay Demonstrating Disruption/Disassembly of Alzheimer's Aβ 1-42Fibrils by PTI-777 and Individual PTI-777 Fractions.

[0107]FIG. 5 is a Color Composite Demonstrating that PTI-777 andIndividual Fractions of PTI-777 Cause of Disruption/Dissolution ofPre-Formed Alzheimer's Aβ Fibrils.

[0108]FIG. 6 is a Black and White Graph of a Circular DichroismSpectroscopy Assay Demonstrating PTI-777 (referred to as PTC12 in thisfigure) Causes a Potent Disruption/Disassembly of B-sheet Structure inAβ 1-42 Fibrils at 3 and 7 days following Incubation.

[0109]FIG. 7 is a Black and White Composite Demonstrating Inhibition ofAlzheimer's Amyloid Fibril Formation as Demonstrated by Negative StainElectron Microscopy.

[0110]FIG. 8 is a graph Demonstrating that PTI-777 Markedly InhibitsAlzheimer's Aβ 1-42 Amyloid Deposition in a Rodent Model of AβFibrillogenesis.

[0111]FIG. 9 is a Color Composite Demonstrating that PTI-777 Causes aMarked Inhibition of Astrocytosis in Brain.

[0112]FIG. 10 are Black and White Graphs Demonstrating the ¹H and¹³C-NMR Profiles of Purified Fraction F of PTI-777. Figure A shows the¹H-NMR profile of purified fraction F in pyridine (d₅) showing 12discrete signals. Figure B demonstrates the ¹³C-NMR profile of purifiedfraction F in pyridine (d₅) showing 16 discrete signal regions

[0113]FIG. 11 is a Black and White Illustration of the ChemicalStructure of Fraction F Identified as Chlorogenic Acid (C₁₆H₁₈O₉;molecular weight 354.31).

[0114]FIG. 12 is a Black and White Composite Demonstrating Successful³H-Labelling of PTI-777 to Produce ³H-PTI-777 to be Used for Assessmentof Blood-Brain-Barrier Penetration Studies.

[0115]FIG. 13 is a Black and White Graph Demonstrating that FollowingIntravenous Administration of ³H-PTI-777 in Adult Sprague-Dawley RatsRadioactivity is Present in Brain Tissue.

[0116]FIG. 14 is a Black and White Graph Demonstrating ElectrosprayTechnique using Time of Flight Mass Spectroscopy of PurifiedPTI-777-Compound J.

[0117]FIG. 15 is a Black and White Graph Demonstrating ElectrosprayTechnique using Fourier Transform of Purified PTI-777-Compound J.

[0118]FIG. 16 is a Black and White Graph Demonstrating Electron Impact(EI) initiated Mass Spectrum of Purified PTI-777-Compound J.

[0119]FIG. 17 is a Black and White Graph Demonstrating Fast AtomBombardment (FAB) Mass Spectroscopy of PTI-777-Compound J that had beenAcetylated.

[0120]FIG. 18 is a Black and White Graph Demonstrating Electron Impact(EI) Mass Spectroscopy of PTI-777-Compound J that had been Acetylated.

[0121]FIG. 19 is a Black and White Graph Demonstrating the ¹H NuclearMagnetic Resonance (NMR) Spectra of PTI-777-Compound J in d₆ Acetone.

[0122]FIG. 20 is a Black and White Graph Demonstrating the ¹³C NMRSpectra of PTI-777-Compound J in d₆ Acetone.

[0123]FIG. 21 is a Black and White Graph Demonstrating the ¹H NMRSpectra of PTI-777-Compound J in D₂O with 0.1% Triflouroacetic acid(TFA).

[0124]FIG. 22 is a Black and White Graph Demonstrating the ¹³C NMRSpectra of PTI-777-Compound J in D₂O with 0.1% Triflouroacetic acid(TFA).

[0125]FIG. 23 is a Black and White Graph Demonstrating CorrelationSpectroscopy (COSEY) of PTI-777-Compound J in d₆ Acetone.

[0126]FIG. 24A is a Black and White Graph Demonstrating CorrelationSpectroscopy (COSEY) of PTI-777-Compound J in acetified D₂O.

[0127]FIG. 24B is a Black and White Graph Demonstrating an Enlargementof the Upper Right Quadrant of the COSEY shown in FIG. 25A.

[0128]FIG. 24C is a Black and White Graph Demonstrating an Enlargementof the Lower Left Quadrant of the COSEY shown in FIG. 25A.

[0129]FIG. 25 is a Black and White Graph Demonstrating ¹H NMR Spectra ofPTI-777-Compound J that had been Acetylated.

[0130]FIG. 26 is a Black and White Graph Demonstrating ¹³C NMR Spectraof PTI-777-Compound J that had been Acetylated.

[0131]FIG. 27 is a Black and White Graph Demonstrating CorrelationSpectroscopy (COSEY) of Pentaacetate Derivative of PTI-777-Compound J.

[0132]FIG. 28A is a Black and White Graph Demonstrating theHeteronuclear Correlation Spectroscopy (HECTOR) of the PentaacetateDerivative of PTI-777-Compound J.

[0133]FIG. 28B is a Black and White Graph Demonstrating an Enlargementof the Aromatic Region of the HECTOR Spectra of the PentaacetateDerivative of PTI-777-Compound J shown in FIG. 29A.

[0134]FIG. 28C is a Black and White Graph Demonstrating an Enlargementof the Methyl Region of the HECTOR Spectra of the PentaacetateDerivative of PTI-777-Compound J shown in FIG. 29A.

[0135]FIG. 29 is a Black and White Graph Demonstrating the UltravioletSpectrum of PTI-777-Compound J.

[0136]FIG. 30 is a Black and White Graph Demonstrating the InfraredSpectra of PTI-777-Compound J.

[0137]FIG. 31 are Black and White Graphs of the Published NMR Spectra ofEpicatechin and Catechin Hydrate.

[0138]FIG. 32 are Black and White Graphs of the Published InfraredSpectra of Epicatechin and Catechin Hydrate.

[0139]FIG. 33 is a Black and White Graph of a Thioflavin T FluorometryAssay used to Determine the Efficacy of “Compound H” and PTI-777 onDisassembly/Dissolution of Pre-Formed Aβ 1-42 Fibrils.

[0140]FIG. 34 is a Black and White Figure of a SDS-PAGE and Western BlotFurther Demonstrating the Disruption of Aβ 1-42 Fibrils, Even inMonomeric Form by “Compound H” and PTI-777.

BEST MODE OF CARRYING OUT THE INVENTION Amyloid and Amyloidosis

[0141] Amyloid is a generic term referring to a group of diverse, butspecific extracellular protein deposits which all have commonmorphological properties, staining characteristics, and x-raydiffraction spectra. Regardless of the nature of the amyloid proteindeposited all amyloids have the following characteristics: 1) anamorphous appearance at the light microscopic level and appeareosinophilic using hematoxylin and eosin stains; 2) all stain with Congored and demonstrate a red/green birefringence as viewed under polarizedlight (Puchtler et al., J. Histochem. Cytochem. 10:355-364, 1962), 3)all contain a predominant beta-pleated sheet secondary structure, and 4)ultrastructurally amyloid usually consist of non-branching fibrils ofindefinite length and with a diameter of 7-10 nm.

[0142] Amyloid today is classified according to the specific amyloidprotein deposited. The amyloid diseases include, but are not limited to,the amyloid associated with Alzheimer's disease, Down's syndrome andHereditary cerebral hemorrhage with amyloidosis of the Dutch type(wherein the specific amyloid is referred to as beta-amyloid protein orAβ), the amyloid associated with chronic inflammation, various forms ofmalignancy and Familial Mediterranean Fever (wherein the specificamyloid is referred to as AA amyloid or inflammation-associatedamyloidosis), the amyloid associated with multiple myeloma and otherB-cell dyscrasias (wherein the specific amyloid is referred to as ALamyloid), the amyloid associated with type II diabetes (wherein thespecific amyloid is referred to as amylin or islet amyloid), the amyloidassociated with the prion diseases including Creutzfeldt-Jakob disease,Gerstmann-Straussler syndrome, kuru and animal scrapie (wherein thespecific amyloid is referred to as PrP amyloid), the amyloid associatedwith long-term hemodialysis and carpal tunnel syndrome (wherein thespecific amyloid is referred to as beta2-microglobulin amyloid), theamyloid associated with senile cardiac amyloid and Familial AmyloidoticPolyneuropathy (wherein the specific amyloid is referred to asprealbumin or transthyretin amyloid), and the amyloid associated withendocrine tumors such as medullary carcinoma of the thyroid (wherein thespecific amyloid is referred to as variants of procalcitonin).

[0143] Although amyloid deposits in clinical conditions share commonphysical properties relating to the presence of a beta-pleated sheetconformation, it is now clear that many different chemical types existand additional ones are likely to be described in the future. It iscurrently thought that there are several common pathogenetic mechanismsthat may be operating in amyloidosis in general. In many cases, acirculating precursor protein may result from overproduction of eitherintact or aberrant molecules (ex. plasma cell dyscrasias), reduceddegradation or excretion (serum amyloid A in some secondary amyloidsyndromes and beta2-microglobulin in long-term hemodialysis), or geneticabnormalities associated with variant proteins (ex. familial amyloidoticpolyneuropathy). Proteolysis of a larger protein precursor moleculeoccurs in many types of amyloidosis, resulting in the production oflower molecular weight fragments that polymerize and assume abeta-pleated sheet conformation as tissue deposits, usually in anextracellular location. What are the precise mechanisms involved, andthe aberrant causes leading to changes in proteolytic processing and/ortranslational modifications is not known in most amyloids.

[0144] Systemic amyloids which include the amyloid associated withchronic inflammation, various forms of malignancy and FamilialMediterranean Fever (i.e. AA amyloid or inflammation-associatedamyloidosis)(Benson and Cohen, Arth. Rheum. 22:36-42, 1979; Kamei et al,Acta Path. Jpn. 32:123-133, 1982; McAdam et al, Lancet 2:572-573, 1975;Metaxas, Kidney Int. 20:676-685, 1981), and the amyloid associated withmultiple myeloma and other B-cell dyscrasias (i.e. AL amyloid)(Harada etal, J. Histochem. Cytochem. 19:1 15, 1971), as examples, are known toinvolve amyloid deposition in a variety of different organs and tissuesgenerally lying outside the central nervous system. Amyloid depositionin these diseases may occur, for example, in liver, heart, spleen,gastrointestinal tract, kidney, skin, and/or lungs (Johnson et al, N.Engl. J. Med. 321:513-518, 1989). For most of these amyloidoses, thereis no apparent cure or effective treatment and the consequences ofamyloid deposition can be detrimental to the patient. For example,amyloid deposition in kidney may lead to renal failure, whereas amyloiddeposition in heart may lead to heart failure. For these patients,amyloid accumulation in systemic organs leads to eventual deathgenerally within 3-5 years. Other amyloidoses may affect a single organor tissue such as observed with the Aβ amyloid deposits found in thebrains of patients with Alzheimer's disease and Down's syndrome: the PrPamyloid deposits found in the brains of patients with Creutzfeldt-Jakobdisease, Gerstmann-Straussler syndrome, and kuru; the islet amyloid(amylin) deposits found in the islets of Langerhans in the pancreas of90% of patients with type II diabetes (Johnson et al, N. Enyl. J. Med.321:513-518, 1989; Lab. Invest. 66:522 535, 1992); thebeta2-microglobulin amyloid deposits in the medial nerve leading tocarpal tunnel syndrome as observed in patients undergoing long-termhemodialysis (Geyjo et al, Biochem. Biophys. Res. Comm. 129:701-706,1985; Kidney Int. 30:385-390, 1986); the prealbumin/ transthyretinamyloid observed in the hearts of patients with senile cardiac amyloid;and the prealbumin/ transthyretin amyloid observed in peripheral nervesof patients who have Familial Amyloidotic Polyneuropathy (Skinner andCohen, Biochem. Bionhys. Res. Comm. 99:1326-1332, 1981; Saraiva et al,J. Lab. Clin. Med. 102:590-603, 1983; J. Clin. Invest. 74:104-119, 1984;Tawara et al, J. Lab. Clin. Med. 98:811-822, 1989).

Alzheimer's Disease and the Aging Population

[0145] Alzheimer's disease is a leading cause of dementia in theelderly, affecting 5-10% of the population over the age of 65 years(1999 Progress Report on Alzheimer's Disease, National Institute onAging/National Institute of Health, Silver Spring, Md.). In Alzheimer'sdisease, the parts of the brain essential for cognitive processes suchas memory, attention, language, and reasoning degenerate, robbingvictims of much that makes us human, including independence. In someinherited forms of Alzheimer's disease, onset is in middle age, but morecommonly, symptoms appear from the mid-60's onward. Alzheimer's diseasetoday affects 4-5 million Americans, with slightly more than half ofthese people receiving care at home, while the others are in manydifferent health care institutions. The prevalence of Alzheimer'sdisease and other dementias doubles every 5 years beyond the age of 65,and recent studies indicate that nearly 50% of all people age 85 andolder have symptoms of Alzheimer's disease (1999 Progress Report onAlzheimer's Disease, National Institute on Aging/National Institute ofHealth, Silver Spring, Md.). 13% (33 million people) of the totalpopulation of the United States is age 65 and older, and this % willclimb to 20% by the year 2025 (1999 Progress Reoort on Alzheimer'sDisease, National Institute on Aging/National Institute of Health,Silver Spring, Md.).

[0146] Alzheimer's disease also puts a heavy economic burden on societyas well. A recent study estimated that the cost of caring for oneAlzheimer's disease patient with severe cognitive impairments at home orin a nursing home is more than $47,000 per year (A Guide toUnderstanding Alzheimer's Disease and Related Disorders, edited by Jorm,New York University Press, New York, 1987). For a disease that can spanfrom 2 to 20 years, the overall cost of Alzheimer's disease to familiesand to society is staggering. The annual economic toll of Alzheimer'sdisease in the United States in terms of health care expenses and lostwages of both patients and their caregivers is estimated at $80 to $100billion (1997 Progress Report on Alzheimer's Disease, National Instituteon Aging/National Institute of Health).

[0147] Tacrine hydrochloride (“Cognex”), the first FDA approved drug forAlzheimer's w disease is a acetylcholinesterase inhibitor (Cutler andSramek, N. Engl. J. Med. 328:808 810, 1993). However, this drug hasshowed limited success in the cognitive improvement in Alzheimer'sdisease patients and initially had major side effects such as livertoxicity. The second more recently FDA approved drug, donepezil (alsoknown as “Aricept”), which is also an acetylcholinesterase inhibitor, ismore effective than tacrine, by demonstrating slight cognitiveimprovement in Alzheimer's disease patients (Barner and Gray, Ann.Pharmacotherapv 32:70-77, 1998; Rogers and Friedhoff, Eur. Neuropsvch.8:67-75, 1998), but is not believed to be a cure. Therefore, it is clearthat there is a need for more effective treatments for Alzheimer'sdisease patients.

Amyloid as a Theralpeutic Target for Alzheimer's Disease

[0148] Alzheimer's disease is characterized by the deposition andaccumulation of a 39-43 amino acid peptide termed the beta-amyloidprotein, Aβ or β/A4 (Glenner and Wong, Biochem. Biophys. Res. Comm.120:885-890, 1984; Masters et al, Proc. Natl. Acad. Sci. USA82:4245-4249, 1985; Husby et al, Bull WHO 71:105-108, 1993). Aβ isderived by protease cleavage from larger precursor proteins termedbeta-amyloid precursor proteins (or βPPs) of which there are severalalternatively spliced variants. The most abundant forms of the βPPsinclude proteins consisting of 695, 751 and 770 amino acids (Tanzi etal, Nature 331:528-530, 1988; Kitaguchi et al, Nature 331:530-532, 1988;Ponte et al, Nature 331:525-527, 1988).

[0149] The small Aβ peptide is a major component which makes up theamyloid deposits of “plaques” in the brains of patients with Alzheimer'sdisease. In addition, Alzheimer's disease is characterized by thepresence of numerous neurofibrillary “tangles”, consisting of pairedhelical filaments which abnormally accumulate in the neuronal cytoplasm(Grundke-Iqbal et al, Proc. Natl. Acad. Sci. USA 83:4913-4917, 1986;Kosik et al, Proc. Natl. Acad. Sci. USA 83:4044-4048, 1986; Lee et al,Science 251:675-678, 1991). The pathologicalhallmarks ofAlzheimer'sdisease is therefore the presence of “plaques” and “tangles”, withamyloid being deposited in the central core of plaques. The other majortype of lesion found in the Alzheimer's disease brain is theaccumulation of amyloid in the walls of blood vessels, both within thebrain parenchyma and in the walls of meningeal vessels which lie outsidethe brain. The amyloid deposits localized to the walls of blood vesselsare referred to as cerebrovascular amyloid or congophilic angiopathy(Mandybur, J. Neuropath. Exp. Neurol. 45:79-90, 1986; Pardridge et al,J. Neurochem. 49:1394-1401, 1987).

[0150] For many years there has been an ongoing scientific debate as tothe importance of “amyloid” in Alzheimer's disease and whether the“plaques” and “tangles” characteristic of this disease, were a cause ormerely the consequences of the disease. Within the last few years,studies now indicate that amyloid is indeed a causative factor forAlzheimer's disease and should not be regarded as merely an innocentbystander. The Alzheimer's Aβ protein in cell culture has been shown tocause degeneration of nerve cells within short periods of time (Pike etal, Br. Res. 563:311-314, 1991; J. Neurochem. 64:253-265, 1995). Studiessuggest that it is the fibrillar structure (consisting of a predominantβ-pleated sheet secondary structure), characteristic of all amyloids,that is responsible for the neurotoxic effects. Aβ has also been foundto be neurotoxic in slice cultures of hippocampus (Harrigan et al,Neurobiol. Aging 16:779-789, 1995) and induces nerve cell death intransgenic mice (Games et al, Nature 373:523-527, 1995; Hsiao et al,Science 274:99-102, 1996). Injection of the Alzheimer's Aβ into ratbrain also causes memory impairment and neuronal dysfunction (Flood etal, Proc. Natl. Acad. Sci. 88:3363-3366, 1991; Br. Res. 663:271-276,1994).

[0151] Probably, the most convincing evidence that Aβ amyloid isdirectly involved in the pathogenesis of Alzheimer's disease comes fromgenetic studies. It has been discovered that the production of Aβ canresult from mutations in the gene encoding, its precursor, beta amyloidprecursor protein (Van Broeckhoven et al, Science 248:1120-1122, 1990;Murrell et al, Science 254:97-99, 1991; Haass et al, Nature Med.1:1291-1296, 1995). The identification of mutations in the beta-amyloidprecursor protein gene which causes early onset familial Alzheimer'sdisease is the strongest argument that amyloid is central to thepathogenetic process underlying this disease. Four reporteddisease-causing mutations have now been discoveredwhich demonstrate theimportance of Aβ in causing familial Alzheimer's disease (reviewed inHardy, Nature Genet. 1:233-234, 1992). All of these studies suggest thatproviding a drug to reduce, eliminate or prevent fibrillar Aβ formation,deposition, accumulation and/or persistence in the brains of humanpatients is believed to serve as an effective therapeutic.

Parkinson's Disease and Alpha-Synuclein Fibril Formation

[0152] Parkinson's disease is a neurodegenerative disorder that ispathologically characterized by the presence of intracytoplasmic Lewybodies (Lewy in Handbuch der Neurolovie, M. Lewandowski, ed., Springer,Berline pp.920-933, 1912; Pollanen et al, J. Neuropath. Exp. Neurol.52:183-191, 1993), the major components of which are filamentsconsisting of alpha-synuclein (Spillantini et al, Proc. Natl. Acad. Sci.USA 95:6469-6473, 1998; Arai et al, Neurosc. Lett. 259:83-86, 1999), an140-amino acid protein (Ueda et al, Proc. Natl. Acad. Sci. USA90:11282-11286, 1993). Two dominant mutations in alpha-synuclein causingfamilial early onset Parkinson's disease have been described suggestingthat Lewy bodies contribute mechanistically to the degeneration ofneurons in Parkinson's disease (Polymeropoulos et al, Science276:2045-2047, 1997; Kruger et al, Nat. Genet. 18:106-108, 1998).Recently, in vitro studies have demonstrated that recombinantalpha-synuclein can indeed form Lewy body-like fibrils (Conway et al,Nature Med. 4:1318-1320, 1998; Hashimoto et al, Brain Res. 799:301-306,1998; Nahri et al, J. Biol. Chem. 274:9843-9846, 1999). Mostimportantly. both Parkinson's disease-linked alpha-synuclein mutationsaccelerate this aggregation process that suggests that such in vitrostudies may have relevance for Parkinson's disease pathogenesis.Alpa-synuclein aggregation and fibril formation fulfills of the criteriaof a nucleation-dependent polymerization process (Wood et al, J. Biol.Chem. 274:19509-19512, 1999). In this regard alpha-synuclein fibrilformation resembles that of Alzheimer's beta-amyloid protein (Aβ)fibrils. Alpha-synuclein recombinant protein, and non-amyloid component(known as NAC-P), which is a 35-amino acid peptide fragment ofalpha-synuclein, both have the ability to form fibrils when incubated at37° C., and are positive with amyloid stains such as Congo red(demonstrating a red/green birefringence when viewed under polarizedlight) and Thioflavin S (demonstrating positive fluorescence) (Hashimotoet al, Brain Res. 799:301-306, 1998; Ueda et al, Proc. Natl. Acad. Sci.USA 90:11282-11286, 1993).

[0153] Parkinson's disease alpha-synuclein fibrils, like the Aβ fibrilsof Alzheimer's disease, also consist of a predominant beta-pleated sheetstructure. Therefore, agents or compounds found to inhibit Alzheimer'sdisease Aβ amyloid fibril formation, are anticipated to also beeffective in the inhibition of alpha-synuclein fibril formation. Theseagents or compounds would therefore also serve as therapeutics forParkinson's disease, in addition to having efficacy as a therapeutic forAlzheimer's disease and other amyloid disorders.

Uncaria tomentosa (Cat's Claw)

[0154] The herb Uncaria tomentosa, also known as “Uña de Gato” (inSpanish) or “Cat's claw” (in English) refers to a woody vine which growswithin the Peruvian Amazon rain forest. This slow growing vine takes 20years to reach maturity, and can grow over 100 feet in length as itattaches and wraps itself around the native trees. It is foundabundantly in the foothills, at elevations of two to eight thousandfeet. The vine is referred to as “Cat's claw” because of its distinctivecurved claw-like thorns which project from the base of its leaves. Thenative Indian tribes traditionally have boiled the inner bark and rootof the herb to make a tea decoction and regard Uncaria tomentosa as asacred medicinal plant. The highly effective properties contained withinthe inner bark of this plant are believed to have a profound andpositive influence on the body, although scientific medical data isgenerally lacking on its potential benefits in humans. The alkaloids andphytochemicals in the inner bark of Uncaria tomentosa are almostidentical to those found in the root, and harvesting this way preservesthe plant and provides for the future of the rainforest.

[0155] Some of the active substances present in Uncaria tomentosa arealkaloids, which occur in the plant and its watery extract as a complexbound to tannins. In this form, only little of them can be activated.The complexes get split by the acid milieu of the stomach; the alkaloidsget transformed into their hydrochloride form, and in this way, get wellabsorbed. A darker Uncaria tomentosa extract means more tannin ispresent and beneficial alkaloids are locked up with the tannins, whichhave formed a non-bioavailable and poorly absorbed complex. A lightgolden color of Uncaria tomentosa suggests that there is less tannins,and more alkaloids available in the extract.

[0156] Besides the presence of alkaloids, Uncaria tomentosa is believedto also contain other beneficial phytochemicals including quinovic acidglycosides, proanthocyanidins, polyphenols, triterpines and the plantsterols beta-sitosterol, stigmasterol and campesterol (P Steinberg“Uncaria tomentosa (Cat's Claw) a wondrous herb from the Peruvian rainforest”, Townsend Letter for Doctors, May, 1994; P. Steinberg, “Cat'sclaw update-Uncaria tomentosa: that wondrous herb from the Peruvian rainforest”, Townstead Letter for Doctors, August/September 1995).

[0157]Uncaria tomentosa is one of the most important plants in the SouthAmerican Peruvian rainforest. A number of oxindole alkaloids havealready been isolated from the inner bark of this plant. Two US patents(U.S. Pat. No. 4,844,901 and U.S. Pat. No. 4,940,725) describe theisolation and use of six oxindole alkaloids from Uncaria tomentosa,which are believed to be “suitable for the unspecified stimulation ofthe immunologic system”. These oxindole alkaloids are believed toprovide a general boost to the immune system as well as have a profoundeffect on the ability of white blood cells and macrophages tophagocytize harmful microorganisms and foreign matter. The mostimmunologically active alkaloid appears to be alloisopteropodine, isomerA, a pentacyclic oxindole alkaloid (U.S. Pat. No. 4,940,725).

[0158] Although some health care providers have suggested that Uncariatomentosa may be used to treat a variety of ailments, nowhere has therebeen any use or suggestion of use, of this compound or extracts thereoffor the treatment of amyloid formation, deposition, accumulation and/orpersistence, such as that which occurs in the amyloidoses, includingAlzheimer's disease and Parkinson's disease. The present inventionclearly demonstrates the effectiveness of Uncaria tomentosa and specificextracts and derivatives thereof obtained from different commercialsources for the 1) inhibition of Alzheimer's Aβ amyloid fibril formation(important for patients in early to mid-stage Alzheimer's disease), and2) causing the dissolution/disruption of preformed Alzheimer's diseaseamyloid fibrils (important for patients in mid-to-late stage Alzheimer'sdisease).

[0159] The following drawings are illustrative of the invention and arenot meant to limit the scope of the invention.

[0160]FIG. 1 is a Black and White Graph of a Thioflavin T FluorometryAssay used to Identify Water-Soluble Fractions of Uncaria tomentosa thatPossess Beta-Amyloid Protein (Aβ) Fibril Disruption Abilities.Thioflavin T fluorometry demonstrates that fractions 13-18 (i.e. 52-72minutes) contain components which disrupt/dissolve pre-formed Aβ 1-40fibrils by 60-75%.

[0161]FIG. 2 is a Preparative HPLC Profile of PTI-777 (Batch II) whichDemonstrates the Major Water-Soluble Amyloid Inhibitory ComponentsIsolated form Uncaria tomentosa. HPLC was monitored at multiplewavelengths using a diode array and a representative profile at 230 nmis shown. Each of the fractions (F through O) were isolated for furtherpurification, and initial structural characterization studies.

[0162]FIG. 3 is a Preparative HPLC Profile of PTI-777 (Batch I) thatDemonstrates the Major Water-Soluble Amyloid Inhibitory ComponentsIsolated from Uncaria tomentosa. HPLC was monitored at multiplewavelengths using a diode array and a representative profile at 230 nmis shown.

[0163]FIG. 4 is a Black and White Graph of a Thioflavin T FluorometryAssay Demonstrating Disruption/Disassembly of Alzheimer's Aβ 1-42Fibrils by PTI-777 and Individual PTI-777 Fractions. Thioflavin Tfluorometry results demonstrate a significant disruption of Aβ 1-42fibrils by PTI-777, PTI-777 individual fractions (including fractions F,G, H, J, K, L, M and N), and to a lesser extent, PTI-00703. PTI-777 wasthe most effective disrupter, whereas Uncaria tomentosa-derived oxindolealkaloids including isopteropodine, pteropodine, isomitraphylline andmitraphylline were ineffective.

[0164]FIG. 5 is a Color Composite Demonstrating that PTI-777 andIndividual Fractions of PTI-777 Cause of Disruption/Dissolution ofPre-Formed Alzheimer's Aβ Fibrils. 125 μM of Aβ 1-42 was incubated at37° C. for 1 week either alone (Figure A), or in the presence of PTI-777(Figure B), Fraction F (Figure C) or Fraction L (Figure D), at anAβ:compound weight ratio of 1:5. Following staining of aliquots withCongo red, a marked reduction in amyloid congophilia (arrows in allfigures; compare to Figure A) as viewed under polarized light wasobserved with PTI-777 (Figure B), and fractions F (Figure C) and L(Figure D).

[0165]FIG. 6 is a Black and White Graph of a Circular DichroismSpectroscopy Assay Demonstrating that PTI-777 (referred to as PTC12 inthis figure) Causes a Potent Disruption/Disassembly of β-sheet Structurein Aβ 1-42 Fibrils at 3 and 7 days following Incubation. 50 μM Aβ 1-42was incubated at 37° C. for 1 week either alone (closed circles), or inthe presence of PTI-777 at an Aβ PTI-777 weight ratio of 1:0.1. At both3 days (open circles) and 7 days (closed triangles), a 85-90%disruption/disassembly of β-sheet (at 220 nm) is shown.

[0166]FIG. 7 is a Black and White Composite Demonstrating Inhibition ofAlzheimer's Amyloid Fibril Formation as Demonstrated by Negative StainElectron Microscopy. 50 μM of Aβ 1-40 incubated for 1 week at 37° C.forms masses of Alzheimer's amyloid fibrils (arrowheads; Figure A). Inthe presence of PTI-777 (at an Aβ:PTI-777 weight ratio of 1:5), onlyamorphous non-fibrillar material is formed (Figure B, arrowheads)demonstrating that PTI-777 inhibits Alzheimer's amyloid fibrilformation.

[0167]FIG. 8 is a graph Demonstrating that PTI-777 Markedly InhibitsAlzheimer's Aβ 1-42 Amyloid Deposition in a Rodent Model of AβFibrillogenesis. 25 μg of Aβ 1-42, 25 μg of Aβ 1-42+PTI-00703, or 25 μgof Aβ 1-42+PTI-777 were directly infused into hippocampus for 1 week inadult Sprague-Dawley rats. The Aβ:PTI-00703 or Aβ:PTI-777 weight ratiowas 1:5. Amyloid deposition in brain was as described previously (Snowet al, Neuron 12:219-234, 1994). The results demonstrated that PTI-777was a much more potent inhibitor of Aβ 1-42 (by ˜26%) fibril depositionthan PTI-00703, indicating that the active ingredients of PTI-00703 werelikely contained within PTI-777.

[0168]FIG. 9 is a Color Composite Demonstrating that PTI-777 Causes aMarked | Inhibition of Astrocytosis in Brain. Tissue sections throughdorsal hippocampus obtained from animals infused for 7 days with 25 μgof Aβ 1-42 in hippocampus (Figures A, B) or Aβ 1-42+PTI-777 (at anAβ:PTI-777 weight ratio of 1:1)(Figures C and D) were immunostained withanti-glial fibrillary acidic protein (GFAP) to assess the degree ofastrocytosis. As expected enhanced GFAP immunostaining (arrows) wasobserved adjacent to the cannula site (marked i) in animals infused withAβ 1-42 only (Figures A and B). On the other hand, animals infused withPTI-777 showed a marked reduction in GFAP immunostaining (Figures C andD), suggestive of a marked inhibition in astrocytosis.

[0169]FIG. 10 are Black and White Graphs Demonstrating the ¹H and¹³C-NMR Profiles of Purified Fraction F of PTI-777. Figure A shows the¹H-NMR profile of purified fraction F in pyridine (d₅) showing 12discrete signals (see text for details). Figure B demonstrates the¹³C-NMR profile of purified fraction F in pyridine (d₅) showing 16discrete signal regions (see text for details).

[0170]FIG. 11 is a Black and White Illustration of the ChemicalStructure of Fraction F that was Identified as Chlorogenic Acid(C₁₆H₁₈O₉; molecular weight 354.31).

[0171]FIG. 12 is a Black and White Composite Demonstrating Successful³H-Labelling of PTI-777 to Produce ³H-PTI-777 to be Used for Assessmentof Blood-Brain-Barrier Penetration Studies. The upper panel demonstratesthe HPLC profile of unlabelled PTI-777 which was monitored at multiplewavelengths using a diode array and a representative profile at 230 nmis shown. The middle profile demonstrates the HPLC profile of ³H-PTI-777at 230 nm. Note that ³H-PTI-777 (middle panel) has nearly the identicalHPLC profile as unlabelled PTI-777 (upper panel) indicating thatlabeling of PTI-777 with ³H did not structurally alter the PTI-777compounds contained within. The lower panel demonstrates theradioactivity distribution (0.5 ml fractions) of ³H-PTI-777 as measuredusing a scintillation counter.

[0172]FIG. 13 is a Black and White Graph Demonstrating that FollowingIntravenous Administration of ³H-PTI-777 in Adult Sprague-Dawley RatsRadioactivity is Present in Brain Tissue. Within 5 minutes ofintravenous injection, ³H-PTI-777 is present in rodent brain tissue, and40% of the radioactivity is maintained in brain over a 24-hour period.This study suggests that PTI-777 or individual components thereof havethe ability to cross the blood-brain-barrier and enter the brain.Therefore, PTI-777 and components thereof have the ability to be used asa therapeutic for Alzheimer's disease and other central nervous systemdisorders.

[0173]FIG. 14 is a Black and White Graph Demonstrating ElectrosprayTechnique using Time of Flight Mass Spectroscopy of PurifiedPTI-777-Compound J.

[0174]FIG. 15 is a Black and White Graph Demonstrating ElectrosprayTechnique using Fourier Transform of Purified PTI-777-Compound J.

[0175]FIG. 16 is a Black and White Graph Demonstrating Electron Impact(EI) initiated Mass Spectrum of Purified PTI-777-Compound J.

[0176]FIG. 17 is a Black and White Graph Demonstrating Fast AtomBombardment (FAB) Mass Spectroscopy of PTI-777-Compound J that had beenAcetylated (i.e. Pentaacetate Derivative of PTI-777-Compound J).

[0177]FIG. 18 is a Black and White Graph Demonstrating Electron Impact(EI) Mass Spectroscopy of PTI-777-Compound J that had been Acetylated(i.e. Pentaacetate Derivative of PTI-777-Compound J).

[0178]FIG. 19 is a Black and White Graph Demonstrating the ¹H NuclearMagnetic Resonance (NMR) Spectra of PTI-777-Compound J in d₆ Acetone.

[0179]FIG. 20 is a Black and White Graph Demonstrating the ¹³C NMRSpectra of PTI-777-Compound J in d₆ Acetone.

[0180]FIG. 21 is a Black and White Graph Demonstrating the ¹H NMRSpectra of PTI-777-Compound J in D₂O with 0.1% Triflouroacetic acid(TFA).

[0181]FIG. 22 is a Black and White Graph Demonstrating the ¹³C NMRSpectra of PTI-777-Compound J in D₂O with 0.1% Triflouroacetic acid(TFA).

[0182]FIG. 23 is a Black and White Graph Demonstrating CorrelationSpectroscopy (COSEY) of PTI-777-Compound J in d₆ Acetone.

[0183]FIG. 24A is a Black and White Graph Demonstrating CorrelationSpectroscopy (COSEY) of PTI-777-Compound J in acetified D₂O.

[0184]FIG. 24B is a Black and White Graph Demonstrating an Enlargementof the Upper Right Quadrant of the COSEY shown in FIG. 25A.

[0185]FIG. 24C is a Black and White Graph Demonstrating an Enlargementof the Lower Left Quadrant of the COSEY shown in FIG. 25A.

[0186]FIG. 25 is a Black and White Graph Demonstrating ¹H NMR Spectra ofPTI-777-Compound J that had been Acetylated (i.e. PentaacetateDerivative of PTI-777-Compound J).

[0187]FIG. 26 is a Black and White Graph Demonstrating ¹³C NMR Spectraof PTI-777-Compound J that had been Acetylated (i.e. PentaacetateDerivative of PTI-777-Compound J).

[0188]FIG. 27 is a Black and White Graph Demonstrating CorrelationSpectroscopy (COSEY) of Pentaacetate Derivative of PTI-777-Compound J.

[0189]FIG. 28A is a Black and White Graph Demonstrating theHeteronuclear Correlation Spectroscopy (HECTOR) of the PentaacetateDerivative of PTI-777-Compound J.

[0190]FIG. 28B is a Black and White Graph Demonstrating an Enlargementof the Aromatic Region of the HECTOR Spectra of the PentaacetateDerivative of PTI-777-Compound J shown in FIG. 29A.

[0191]FIG. 28C is a Black and White Graph Demonstrating an Enlargementof the Methyl Region of the HECTOR Spectra of the PentaacetateDerivative of PTI-777-Compound J shown in FIG. 29A.

[0192]FIG. 29 is a Black and White Graph Demonstrating the UltravioletSpectrum of PTI-777-Compound J.

[0193]FIG. 30 is a Black and White Graph Demonstrating the InfraredSpectra of PTI-777-Compound J.

[0194]FIG. 31 are Black and White Graphs of the Published NMR Spectra ofEpicatechin and Catechin Hydrate.

[0195]FIG. 32 are Black and White Graphs of the Published InfraredSpectra of Epicatechin and Catechin Hydrate.

[0196]FIG. 33 is a Black and White Graph of a Thioflavin TFluorometryAssay used to Determine the Efficacy of “Compound H” andPTI-777 on Disassembly/Dissolution of Pre-Formed Aβ 1-42 Fibrils. Both“compound H: and PTI-777 cause a potent disassembly/disruption ofpre-formed Aβ 1-42 fibrils.

[0197]FIG. 34 is a Black and White Figure of a SDS-PAGE and Western BlotFurther Demonstrating the Disruption of Aβ 1-42 Fibrils, Even inMonomeric Form by “Compound H” and PTI-777. 25 μM of pre-fibrillized Aβ1-42 was incubated at 37° C. for 1 week either alone or in the presenceof PTI-777 (1:1 wt/wt ratio), “compound H” (1:1 wt/wt ratio) or EDTA(1:1 wt/wt ratio). Following SDS-PAGE, Aβ was detected by ECL usinganti-6E10 antibody. Aβ 1-42 monomers were not detected following theincubation of Aβ 1-42 with either PTI-777 or “compound H” suggestingthat both were capable of causing a disappearance of monomeric Aβ 1-42.

EXAMPLES

[0198] The following examples are put forth so as to provide those withordinary skill in the art with the disclosure and description of thecomposition, methods of isolation and use of amyloid inhibitingcompounds derived from Uncaria tomentosa and related plants to inhibitamyloid fibril formation, and cause dissolution/disruption of preformedamyloid fibrils. However, it should not be construed that the inventionis limited to these specific examples.

Example 1 Initial Isolation and Testing of Active Ingredients Derivedfrom Uncaria tomentosa

[0199] Assay-guided affinity fractionation and high pressure liquidchromatography (HPLC) was used to separate and purify the major Aβamyloid inhibitory active components present in Uncaria tomentosa.Several different separation techniques were evaluated to determine thebest methodology to purify and test the active Aβ amyloid inhibitingcomponents of Uncaria tomentosa. Initial studies utilized water extractsof Uncaria tomentosa applied to different Affi-Prep 10 gel columnsderivatized with Tris-HCl, ammonia or ethanolamine. Methanol, ethanol oracetonitrile were found to effectively elute the Uncaria tomentosaactive components from the column. It was also discovered that aTris-HCl derivatized column was most effective in binding the Aβ amyloidinhibitory components of Uncaria tomentosa which suggested that theactive ingredients may have affinity for both hydroxy (due to the Tris)and hydrophobic (due to the resin used) groups. In order to scale-upseparation of the active components of Uncaria tomentosa, aTris-acrylate column was prepared by rinsing 25 ml of Affi-Prep 10 gel(Biorad) with distilled water and incubating with 100 ml Tris-HCl (1 M,pH 8.0). The resulting material was packed into a 20 ml column (MT20,Biorad), attached to a 1100 series Hewlett Packard HPLC with diode arraydetector. The column was equilibrated at a flow rate of 0.5 ml/min withwater. A water soluble extract prepared from 400 mg of lyophilizedUncaria tomentosa in 2 ml of distilled water was injected onto theTris-Derivatized Affi-gel 10 column and eluted using the followingprofile: 0-10 min 100% water; 10-100 min, 0-100% acetonitrile, and100-110 min, 100% acetonitrile. Fractions were collected every 4minutes. Aliquots from fractions 1-22 (i.e. 4 mins to 84 mins) were thenincubated with fibrillar Aβ 1-40 (FIG. 1) or Aβ 1-42 (not shown) for 2hours (at a wt/wt ratio of 1:1) and tested for their ability todisrupt/disassemble pre-formed Aβ fibrils using Thioflavin T fluorometryas previously described (Castillo et al, J. Neuroscience 69:2452-2465,1997). As shown in FIG. 1, fibrillar Aβ 1-40 alone demonstrated afluorescence of 836 +/−61 fluorescence units. Fractions 13-18 (i.e.52-72 mins) demonstrated the greatest ability (from 60-75%) todisrupt/disassemble pre-formed Aβ 1-40 fibrils, as indicated by a markedlowering of fluorescence (FIG. 1). Similar results were obtained withpre-formed Aβ 1-42 fibrils (not shown). This study suggested that themost active Aβ amyloid inhibitory components present within the watersoluble extract of Uncaria tomentosa (i.e. PTI-00703) were locatedwithin fractions 13-18.

Example 2 Purification of the Major Amyloid Inhibitory Components in theWater-Soluble Fraction of Uncaria tomentosa (PTI-777 Batch II Protocol)

[0200] Since our initial studies suggested that the PTI-00703 activityretained on the Tris-acrylate column may be due to the presence ofhydroxy and hydrophobic groups, we implemented the use of a LH20 column(Pharmacia) as an initial column for larger scale preparations. LH20 isa carbohydrate-based resin derivatized in such a way that it acquireshydrophobic characteristics, and contains both hydroxy (fromcarbohydrates) and hydrophobic groups (from derivatization). When thewater extract of Uncaria tomentosa (10 gms in 200 ml) was applied to aLH20 column, 100% of the Aβ amyloid inhibitory activity was retained bythe column even after washing with 4 volumes of water. However, the Aβamyloid inhibitory activity could be eluted with 3 volumes of 100%methanol. Materials retained on the column even following the 100%methanol wash were believed to primarily consist of tannic acids aspreviously described (Haegerman and Klucher, In: Plant Flavanoids inBiology and Medicine: Biochemical, Pharmacoloical and Structure-ActivityRelationships, Edited by V Cody, E. Middelton Jr., J. Harborne, Alan R.Liss, New York, 1986, pp.67-76). These tannin materials were latereluted with a 70% (v/v) acetone-water mixture applied during the columnclean-up step. We found that fractions 13-18 (as described above), whichconsist of the major water-soluble active Aβ amyloid inhibitorycomponents of Uncaria tomentosa, demonstrated similar HPLC profiles tothe 100% methanol eluate of LH20. This finding indicated that regardlessof whether a LH20 column or a Tris-HCl derivatized Affigel-10 column wasused, similar components could be recovered. These components arecollectively referred to as “PTI-777”.

[0201] In our preparative protocol for isolation, purification andtesting of the main active water-soluble components within Uncariatomentosa, we scaled up use of the LH20 column for preparative purposes.To that end we used the following methodology for the extraction of“PTI-777 (Batch II)” (Table 1 outlines the protocol). 1 kg of Uncariatomentosa was extracted using a 5 liter polypropylene container to which4000 ml of methanol was added. Following mixing with a Barnant mixer,the extract was centrifuged at 2,500×g using a Beckman GS-6KR centrifugefor 30 minutes and the supernatant was collected. The insoluble materialwas extracted 3 more times in a similar manner and the combinedsupernatants (containing the active ingredients) were evaporated todryness (or until 500 ml volume is reached) using a rotary evaporator at50° C. (yield was 100 grams=˜10% of starting material). The powderedextract (or 500 ml volume) was then washed 4 times with 300 ml ofpetroleum ether (to remove any lipids), and the ether layer wasdiscarded. The methanol was then evaporated to dryness using a rotaryevaporator at 50° C. The solid material was then extracted 5 times with150 ml of distilled water. Each extraction was followed bycentrifugation at 2,500× for 30 minutes. The combined supernatants(volume ˜750 ml) were then lyophilized using a freeze-dryer. Theresulting lyophilized water extract (yield was 50 grams=˜5% of startingmaterial) was dissolved in 500 ml of distilled water, and 50-100 mlportions (˜3 grams) were applied to a 400 ml LH-20 column equilibratedwith distilled water. The LH-20 column was then eluted with 1,100 ml ofdistilled water (˜3 column volumes) and the amber/yellow, non-activefractions were discarded. The LH-20 column was then eluted with 1100 mlof 100% methanol (˜3 column volumes) and a set of active fractions werecollected and evaporated to dryness using a rotary evaporator. 12 gramsof powdered extract was obtained (1.2% of the starting Uncaria tomentosamaterial) which was referred to as “PTI-777 (Batch II)”.

[0202] Prior to additional purification of PTI-777 Batch II by reversephase (C18) HPLC, the material was dissolved in distilled water (80mg/ml) and applied 5 ml at a time to a 10 gm disposable C18 SPE columnequilibrated in 95% water/5% acetonitrile/0.1% TFA (referred to assolvent A). The column was washed with 3 column bed volumes of solvent Aand the eluate was discarded (which contained inactive components). Thecolumn was eluted with 3 column bed volumes of solvent A containing12.5% solvent B (solvent B=95% acetonitrile/5% water/0.1% TFA). Thesefractions containing the Aβ amyloid inhibitory components were thenlyophilized for further purification and analysis.

[0203] In an effort to isolate individual compounds responsible for theAβ amyloid inhibitory activity of PTI-777 Batch II, preparative HPLC wasused. For these studies, 50 mg of PTI-777 Batch II (prepared asdescribed above) was injected multiple times into a Hewlett-Packard 1100Series HPLC instrument with diode array detector, fitted with a 2.2cm×25 cm Vydac 218TP1022 C18 reverse-phase column maintained at 25° C.and at a flow rate of 5 ml/min. The sample was eluted with the followingsolvent profile: 10% B for 0-20 mins., 10-100% B gradient for 20-30mins., and 100-10% B gradient from 30-31 mins. where A=95% water/5%acetonitrile/0.1% TFA, and B=95% acetonitrile/5% water/0.1%TFA. Underthese conditions, PTI-777 Batch II separated into 11 major components asrevealed by uv/vis detection (diode array). Fractions containing 11components were collected and labeled as follows: fraction G (16-17mins), fraction F (17-18 mins), fraction H (19-20 mins), fraction I (21mins), fraction J (22-23 mins), fraction K₁ (24 mins), fraction K₂ (25mins), fraction L (26-27 mins), fraction M (28 mins), fraction N (29-30mins) and fraction O (32-33 mins). The HPLC profile of PTI-777 Batch IIis shown in FIG. 2. TABLE 1 PTI-777 Batch II Isolation Protocol Step 1 1kg of Uncaria tomentosa bark powder + 4000 ml methanol (mix) Step 2Centrifuge for 30 minutes at 2,500 Xg Step 3 Collect supernatant -repeat centrifugation and supernatant collection steps 4 times Step 4Evaporate to dryness or until volume is 500 ml at 50° C. Step 5 Washwith 300 ml of petroleum ether and discard ether layer (repeat 4 times)Step 6 Evaporate to dryness at 50° C. (100 g or ˜10% of startingmaterials). Extract with 150 ml of distilled water, followed bycentrifugation for 30 minutes at 2,500 Xg (repeat 5 times) Step 7Lyophilize water extract (yield ˜50 grams = ˜5% of starting material)Step 8 Dissolve 50 g lyophilized water extract in 500 ml distilled waterand apply 50-100 ml at a time on 400 ml LH-20 equilibrated with waterStep 9 Elute with 1100 ml distilled water and discard Step 10 Elute with1100 ml methanol, collect fractions and evaporate to dryness (thesefractions contain mostly G, F, H, J, K₁, K₂, L, M, N, O and other morehydrophobic fractions) (yield ˜12 grams = ˜1.2% of starting material;these fractions are the most active against Aβ amyloid fibrillogenesis)Step 11 Clean up fractions obtained from step 10 as follows. Dissolve inwater (80 mg/ml) and apply 5 ml at a time to a 10 gm of disposable C18SPE column equilibrated in solvent A and wash with 3 volumes of solventA and discard the eluate. Elute the clean fraction with 3 volumes ofsolvent A containing 12.5% solvent B. Lyophylize the correspondingfractions (˜10 g from 1 kg). Where Solvent A = 95% water/5%acetonitrile/0.1% TFA, and Solvent B = 95% acetonitrile/5% water/0.1%TFA. Step 12 Fractionate fractions from step 11 on HPLC using 90 ml C18reverse-phase HPLC column to isolate individual components (i.e.fractions F, G, H, I, J, K₁, K₂, L, M/N & O)

[0204] Conditions; 50 mg/ml injections (in solvent A); 40 times; 5mls/min flow rate; collect 5 ml fractions every 1 minute

[0205] Gradient=10% B 0-20 minutes, 10-100%B20-30 minutes, 100-10%B30-31minutes

[0206] Run time=35 minutes

[0207] Solvent A=95% water/5%acetonitrile/0.1% TFA

[0208] Solvent B=95% acetonitrile/5% water/0.1% TFA Retention Times:Fraction G 16-17 minutes Fraction K₂ 25 minutes Fraction F 17-18 minutesFraction L 26-27 minutes Fraction H 19-20 minutes Fraction M 28 minutesFraction I 21 minutes Fraction N 29-30 minutes Fraction J 22-23 minutesFraction O 32-33 minutes Fraction K₁ 24 minutes

Example 3 Modification of the PTI-777 Protocol to Reduce Fractions F, G,M, N and O (PTI-777 Batch I Protocol)

[0209] As outlined in Table 2, a second modified protocol (as describedbelow) was also used to isolate the water-soluble amyloid inhibitorycomponents from Uncaria tomentosa, and this protocol isolated a set ofactive fractions referred to as PTI-777 Batch I. PTI-777 Batch Icomponents are identical to PTI-777 Batch II components, however, thereis lesser amounts of fractions F, G, M, N, and O which are the yellowishcomponents. Our data demonstrate that PTI-777 Batch I contains slightlymore amyloid inhibitory activity than PTI-777 Batch II. For theisolation of PTI-777 Batch I the following methodology was used: 1 kg ofUncaria tomentosa was extracted using a 5 liter polypropylene containerto which 4000 ml of methanol was added. Following mixing with a Barnantmixer, the extract was centrifuged at 2,500× g using a Beckman GS-6KRcentrifuge for 30 minutes and the supernatant was collected. Theinsoluble material was extracted 3 more times in a similar manner andthe combined supernatants (containing the active ingredients) wereevaporated to dryness (or until volume is ˜500 ml) using a rotaryevaporator at 50° C. (yield was 100 grams=˜10% of starting material).The powdered extract (or 500 ml volume) was then washed 4 times with 300ml of petroleum ether (to remove any lipids), and the ether layer wasdiscarded. The material was then evaporated to dryness using a rotaryevaporator at 50° C. The solid material was then extracted 5 times with150 ml of distilled water. Each extraction was followed bycentrifugation at 2,500× for 30 minutes. The combined supernatants(volume ˜750 ml) were then lyophilized using a freeze-dryer. Theresulting lyophilized water extract (yield was 50 grams=˜5% of startingmaterial) was dissolved in 500 ml of distilled water, and 50-100 mlportions (˜3 grams) were applied to a 400 ml LH-20 column equilibratedwith distilled water. The LH-20 column was then eluted with 1,100 ml ofdistilled water (3 column volumes) and the amber/yellow, non-activefractions were discarded. The LH-20 column was then eluted with 1100 mlof 50% methanol/water and yellowish fractions were collected whichcontained mostly fractions F, G, M, N & O (yield was 6 grams=˜0.6% ofstarting material). This added step separated away some of the lessactive components of PTI-777. The LH-20 column was then eluted with 1100ml of 100% methanol (˜3 column volumes) and a set of active fractionswere collected (these fractions contained mostly H. J, K₁, K₂ and L) andevaporated to dryness using a rotary evaporator. 6 grams of powderedextract was obtained (0.6% of the starting Uncaria tomentosa material)which was referred to as “PTI-777 (Batch I)”.

[0210] Prior to additional purification of PTI-777 Batch I by reversephase (C18) HPLC, the material was dissolved in distilled water (80mg/ml) and applied 5 ml at a time to a 10 gm disposable C18 SPE columnequilibrated in 95% water/5% acetonitrile/0.1% TFA (referred to assolvent A). The column was washed with 3 volumes of solvent A and theeluate was discarded (which contained inactive components). The columnwas eluted with 3 volumes of solvent A containing 12.5% solvent B(solvent B=95% acetonitrile/5% water/0.1% TFA). These fractionscontaining the Aβ amyloid inhibitory components were then lyophilizedfor further purification and analysis.

[0211] As an alternative to the use of C18 SPE columns the PTI-777material can also be further purified by flash chromatographytechniques. As one example, 5 grams of post LH-20 lyophilized materialcontaining PTI-777 was dissolved with 20 ml of distilled water. Thesolubilized solution was then transferred into a 50 ml disposablecentrufuge tube and spun for 15 minutes on a centrifuge at ˜6,000 ×g. AVarian Chromozone apparatus (set at about 85 psi) was then used at aflow rate setting of level 5, and then the column was equilibrated with500 ml of solvent A (solvent A=95% water/5% acetonitrile/0.1% TFA). A 20ml solution of the PTI-777 solublized material was loaded into a syringeand inserted into the sample port and loaded. With the flow rate settingat 5 or 6, the column was then washed with 500 ml of solvent A(equivalent to 3 column bed volumes) and the eluate which does notcontain amyloid inhibitory activity, was discarded. The clean fractioncontaining the PTI-777 active fractions were then eluted with 550 ml ofsolvent A (equivalent to close to 3 column bed volumes) containing 12%solvent B (solvent B=95% acetonitrile/5% water/0.1% TFA). The first 50ml of the eluate was discarded. The PTI-777 fraction was collected intoa 1 liter pyrex bottle for further purification as described below.

[0212] In an effort to isolate individual compounds responsible for theAβ amyloid inhibitory activity of PTI-777 Batch I, preparative HPLC wasused. For these studies, 50 mg of PTI-777 Batch I (prepared by methodsas described above) was injected multiple times into a Hewlett-Packard1100 Series HPLC instrument with diode array detector, fitted with a 2.2cm×25 cm Vydac 218TP1022 C18 reverse-phase column (95 ml) maintained at25° C. and at a flow rate of 5 ml/min. The sample was eluted with thefollowing solvent profile: 10% B for 0-20 mins., 10-100% B gradient for20-30 mins., and 100-10% B gradient from 30-31 mins. where A=95%water/5% acetonitrile/0.1% TFA, and B=95% acetonitrile/5% water/0.1%TFA.Under these conditions, PTI-777 Batch I separated into components (FIG.5), as revealed by uv/vis detection (diode array). These fractions werecollected and labeled as follows: fraction G (13-14 mins), fraction F(15-16 mins), fraction H (17-20 mins), fraction 1 (21 mins), fraction J(22-23 mins), fraction K₁ (24 mins), fraction K₂ (25 mins), fraction L(26-27 mins), fraction M (27-28 mins), and fraction N (28-29 mins). Thismodification led to the reduction of fractions F, G, M, N and O. TheHPLC profile of PTI-777 Batch I is shown in FIG. 3. TABLE 2 PTI-777Batch I Isolation Protocol Step 1 1 kg of Uncaria tomentosa barkpowder + 4000 ml methanol (mix) Step 2 Centrifuge for 30 minutes at2,500 Xg Step 3 Collect supernatant - repeat centrifugation andsupernatant collection steps 4 times Step 4 Evaporate to dryness oruntil volume is 500 ml at 50° C. Step 5 Wash with 300 ml of petroleumether and discard ether layer (repeat 4 times) Step 6 Evaporate todryness at 50° C. (100 g or ˜10% of starting materials). Extract with150 ml of distilled water, followed by centrifugation for 30 minutes at2,500 Xg (repeat 5 times) Step 7 Lyophilize water extract (yield ˜50grams = ˜5% of starting material) Step 8 Dissolve 50 g lyophilized waterextract in 500 ml water and apply 50-100 ml at a time on 400 ml LH-20equilibrated with water Step 9 Elute with 1100 ml water and discard Step9A Elute with 1100 of 50% methanol/distilled water and collect fractions(these fractions contain mostly F, G, M and N) (yield ˜6 grams = ˜0.6%of starting material) Step 10 Elute with 1100 ml methanol and collectfractions (these fractions contain mostly H, J, K₁, K₂, L and other morehydrophobic fractions) (yield ˜6 gram = ˜0.6% of starting material;these fractions are the most active against Aβ amyloid fibrillogenesis)Step 11 Clean up fractions obtained from steps 9a and 10 as follows.Dissolve in water (80 mg/ml) and apply 5 ml at a time to a 10 gm ofdisposable C18 SPE column equilibrated in solvent A and wash with 3volumes of solvent A and discard the eluate. Elute the clean fractionwith 3 volumes of solvent A containing 12.5% solvent B. Lyophylize thecorresponding fractions (˜5 g each obtained from steps 9A and 10).Where, Solvent A = 95% water/5% acetonitrile/ 0.1% TFA, and Solvent B =95% acetonitrile/5% water/0.1% TFA. Step 12 Fractionate fractions fromstep 11 (which consists of two separate fractions) on HPLC using 90 mlC18 reverse-phase HPLC column to isolate individual components.

[0213] Conditions; 50 mg/ml (in solvent A) injections; 40 times; 5mls/min flow rate; collect 5 ml fractions every 1 minute

[0214] Gradient=10% B 0-20 minutes, 10-100%B20-30 minutes, 100-10%B30-31minutes

[0215] Run time=35 minutes

[0216] Solvent A=95% water/5%acetonitrile/0.1% TFA

[0217] Solvent B=95% acetonitrile/5% water/0.1% TFA Retention Times:Fraction G 13-14 minutes Fraction K₁ 24 minutes Fraction F 15-16 minutesFraction K₂ 25 minutes Fraction H 17-20 minutes Fraction L 26-27 minutesFraction I 21 minutes Fraction M 27-28 minutes Fraction J 22-23 minutesFraction N 28-29 minutes

Example 4 Analytical/Semi-Preparative Fractionation

[0218] Fractions from step 11 (see Table 2 above) can also be analyzedby HPLC using a C18 reverse-phase HPLC column (10×250 mm; 20 ml resin)to fractionate the various components including fractions F, G, H, I, J,K₁, K₂, L, M/N and O, as well as more hydrophobic components. Usingthese conditions ˜50 μg in solvent A was fractionated using the HPLCapparatus at a flow rate of ˜1.5 ml/minute. The gradient run was 10% Bfrom 0 to 20 minutes, 10% -100% B gradient from 20 to 30 minutes, and100% -10% B gradient from 30 to 31 minutes. Solvent B=95%acetonitrile/5% water/0.1% TFA. Using this method, the retention timesof the various fractions were as follows: Fraction G (12-13 minutes),Fraction F (13-14 minutes), Fraction H (15 minutes), Fraction I (16minutes), Fraction J (18-19 minutes), Fraction K₁ (20 minutes), FractionK₂ (21 minutes), Fraction L (21-23 minutes), Fraction M (23 minutes),Fraction N (24 minutes) and Fraction O (26-27 minutes).

Example 5 Scale-Up Procedures for the Isolation of the AmyloidInhibitory Components of Uncaria tomentosa

[0219] Based on our work 1 kg of Uncaria tomentosa (i.e. Cat's claw)bark powder is expected to yield 300-500 mg of each of the purifiedamyloid inhibitory components from PTI-777 (i.e. fractions G through O).Scale up procedures may be utilized for the isolation of PTI-777 and itsindividual components. For these procedures the Uncaria tomentosa barkpowder can be extracted in 1 kg batches as described in detail inExamples 2 and 3. The only difference in procedure is that thecentrifugation steps can take place at higher speeds/centrifugationforce (i.e. ×20,000 g instead of ×2,500 g) using a Sorvall Discovery100S or equivalent ultracentrifuge. Methanol eluted fractions from theLH-20 column are collected and will contain the PTI-777 fractions. Priorto preparative HPLC, the PTI-777 containing fractions (80 mg/ml) areapplied 5 ml at a time to a 10 gm disposable C18 SPE column equilibratedin 95% water/5% acetonitrile/0.1% TFA (i.e. solvent A). The column isthen washed with 3 volumes of solvent A and the resulting eluatediscarded (contains remaining inactive components). The materialcontaining PTI-777 is then eluted from the column with 3 volumes ofsolvent mixture comprising 87.5% A and 12.5% solvent B (solventB=95%acetonitrile/5% water/0.1% TFA) and lyophilized prior to furtherpurification and analysis.

[0220] Preparative HPLC is used to separate the PTI-777 mixture andobtain pure samples of the compounds isolated from the mixture. Forfaster and scale-up preparations, samples of PTI-777 (1 g in 5-10 ml insolvent A) are injected onto a 4.14 cm×25 cm Varian Dynamax C-18 reversephase column fitted to a Varian Prostar 215 solvent delivery system, anda Varian model 320 TV-Vis detector. The separation is carried out atambient temperature, a flow rate of 50 ml/min, and UV detection at 230nm. The solvent gradient profile is as follows:0-4 min, 25%B; 4-11 min,25-30%B; 11-14 min, 30-90% B; 14-17 min, 90% B; and 17-19 min, 90-25% B;where A is distilled water with 0.1% TFA and B is methanol with 0.1%TFA. Based on our work, these modified HPLC conditions will also resultin the separation and purification of the 11 major fractions (G-O incl.K₁ and K₂) previously isolated from PTI-777. The fractions obtainedunder these procedures can be correlated with the original fractions byHPLC under conditions described in the Examples above. Finalpurification of PTI-777 individual components within each fraction mayrequire additional HPLC to separate each of the major compounds withineach fraction, from any minor components that may be present. The majorcomponents of each fraction (which usually represent 90% of thematerial) are isolated by the pooling (and drying) of fractionscomprised of a single major peak when viewed at 210 nm on HPLC. Theresulting pure material can be used for in vitro/in vivo testing andstructural elucidation as described herein. To assess the purity ofindividual major PTI-777 components HPLC/diode array detection is usedas well as mass spectroscopy and nuclear magnetic resonance (NMR)spectroscopy.

Example 6 In Vitro Testing of Individual Fractions within PTI-777 for AβAmyloid Inhibitory Activity

[0221] The bioactivities of PTI-777 (either derived from Batch I orBatch II) and its isolated individual fractions (i.e. fractions Fthrough O) were evaluated in a number of different in vitro assays.Testing included the use of Thioflavin T fluorometry, Congo red stainingassays, solid phase binding immunoassays (for analysis of inhibition ofAβ-Aβ and Aβ-glycosaminoglycan interactions) and negative stain electronmicroscopy to assess the effects on inhibition of Aβ fibril formationand growth, interactions with specific glycosaminoglycans, as well asability to disrupt/disassemble pre-formed Aβ fibrils. In mostexperiments, individual isolated fractions of PTI-777 were directlycompared to a water extract of Uncaria tomentosa (i.e. PTI-00703),PTI-777 (containing the mixture of fractions F through O), and the majoroxindole alkaloids isolated from Uncaria tomentosa and thought tocontain important bioactivity as previously described in two US patents(U.S. Pat. No. 4,844,901 and U.S. Pat. No. 4,940,725). These oxindolealkaloids are believed to provide a general boost to the immune systemas well as have a profound effect on the ability of white blood cellsand macrophages to phagocytize harmful microorganisms and foreign matter(U.S. Pat. No. 4,940,725). The Uncaria tomentosa oxindole alkaloidstested in these experiments (described below) included isopteropodine,pteropodine, isomitraphylline and mitraphylline. Access to thesealkaloids also allowed us to use them as markers to see if they werepresent in our bioactive extracts and fractions of PTI-777.

Example 7 Inhibition of Aβ Fibrillogenesis by PTI-777 and PTI-777Individual Fractions

[0222] In one set of studies, Thioflavin T fluorometry and Congo redstaining assays were used to compare the ability of PTI-777, PTI-777individual fractions (including fractions F, G, H, J, K, L, M and N),PTI-00703, and alkaloids isolated from Uncaria tomentosa, to cause adisruption/disassembly of pre-formed Aβ 1-42 fibrils. As shown in FIG.4, the results of 5 different Thioflavin T fluorometry experimentsindicated that PTI-00703 caused a significant 53+/−2.5% disruption ofpre-formed Aβ 1-42 fibrils. On the other hand, individual PTI-777fractions including fraction F (64.0+/−1.7% inhibition), fraction G(62.3+/−8.5% inhibition), fraction H (56.3+/−2.1% inhibition), fractionJ (68.7+/−2.0% inhibition), fraction K (which consisted of both K₁ andK₂; 58.0 +/−4.6% inhibition), fraction L (68.3+/−2.3% inhibition),fraction M (64.0+/−1.5% inhibition) and fraction N (63.0+/−1.0%inhibition) were all similarly quite effective in causing a significantdisruption/ disassembly of pre-formed Aβ 1-42 fibrils. Surprisingly,PTI-777 (i.e. mixture of fractions F through O) was a significantly moreeffective disrupter of Aβ 1-42 fibrils (by 87.3+/−3.0%) than any of theindividual fractions tested. The alkaloids isolated from Uncariatomentosa (isopteropodine, pteropodine, isomitraphylline andmitraphylline) had little or no effect on disruption/ disassembly ofpre-formed Aβ 1-42 fibrils. These studies indicated that PTI-777 and itsindividual fractions were more effective Aβ amyloid disrupters, thanPTI-00703 alone. In addition, it was evident that the combination offractions such as observed with PTI-777 (a mixture of 7-11 majorcomponents) were even more active than any of the individual PTI-777fractions alone, suggesting a possible synergistic effect betweendifferent PTI-777 components. Lastly, the fact that isolated alkaloidsfrom Cat's claw (i.e. Uncaria tomentosa) were basically ineffective inthe disruption of pre-formed Aβ 1-42 fibrils, suggested that oxindolealkaloids were not likely responsible for the Aβ amyloid inhibitoryeffects exerted by PTI-777, the individual PTI-777 fractions testedabove, and PTI-00703. It is likely that compounds not previouslyisolated and identified from Uncaria tomentosa were actually responsiblefor the observed anti-Aβ amyloid inhibitory effects.

[0223] Congo red staining assay experiments confirmed the results of theThioflavin T fluorometry studies, and revealed the efficacy of PTI-777and individual PTI-777 fractions on their ability to disrupt Alzheimer'sAβ 1-40 and Aβ 1-42 pre-formed fibrils, as well as to inhibit Aβ 1-40fibril formation. In these experiments, 125 μM of Aβ 1-40 or Aβ 1-42 wasincubated at 37° C. for 1 weekin TBS either alone, or in the presence ofincreasing amounts of PTI-777, individual PTI-777 fractions isolatedfrom HPLC, or PTI-00703. At 1, 3 and 7 days, 10 μl aliquots were taken,put on glass slides, air-dried, stained with Congo red and viewed underpolarized light. Usually testing of Aβ:PTI-777 or individual PTI-777fractions were at an Aβ:compound weight ratio of 1:1 or 1:5. An exampleof the results of one of these experiments is shown in FIG. 5. In thisparticular experiment, Aβ 1-42 was incubated either alone, or in thepresence of PTI-777, fractions F, G, H, J, K (combined K₁ and K₂), L, Mand N at 37° C. for 1 week at an Aβ:compound weight ratio of 1:5. Asshown in FIG. 5A, Aβ 1-42 alone at 7 days displayed extensive amyloidcongophilia (i.e. red/green birefringence) when viewed under polarizedlight indicative of massive amounts of amyloid fibrils (Puchtler et al,J. Histochem. Cytochem. 10:355-364, 1962). In contrast, Aβ 1-42 in thepresence of PTI-777 (FIG. 5B), or other individual PTI-777 fractionsincluding fraction F (FIG. 5C), G, H, J, K (not shown), L (FIG. 5D), Mand N (not shown), all displayed a marked reduction in amyloidcongophilia indicative of an inhibition of Aβ amyloid fibril formation.

Example 8 Disruption of β-Sheet Structure in Aβ 1-42 Fibrils by PTI-777as Demonstrated by Circular Dichroism Spectroscopy

[0224] In this study the effects of PTI-777 on potentialdisruption/disassembly of β-pleated sheet structure in Aβ 1-42 fibrilswas determined by circular dichroism (CD) spectroscopy. 50 μM Aβ 1-42(Bachem Inc) was incubated at 37° C. for 1 week in phosphate-bufferedsaline (pH 7.4) either alone or in the presence of PTI-777 (referred toas PTC12 in FIG. 6) at an Af:PTI-777 weight ratio of 1:0.1. CD spectrawere collected at 25° C. on an AVIV CD Spectrometer 62DS. Measurementswere carried out in a 0.5 mm path length quartz cuvette, over the rangeof 190-260 nm. The instrument was calibrated with an aqueous solution of(+)-10-camphorsulfonic acid. CD spectra consisted of an average of aseries of scans made at 0.5 nm intervals. As shown in FIG. 6, Aβ 1-42 at3 days (closed circles) and at 7 days (not shown) demonstrated acharacteristic pattern of extensive β-pleated sheet structure as shownby the curve and minima at ˜220 nm. In the presence of PTI-777 at both 3days (open circles) and 7 days (closed triangles) an 85-90%disruption/disassembly of β-pleated sheet was observed as shown by asmoothing of the curve especially at 220 nm. This study indicated thatPTI-777 is a potent disrupter of the β-sheet structure characteristic ofAlzheimer's amyloid fibrils.

Example 9 Inhibition ofAlzheimer's Aβ Fibril Formation by PTI-777 asDemonstrated by Negative Stain Electron Microscopy

[0225] The potent inhibition of Aβ 1-40 fibril formation by PTI-777, andto a lesser extent PTI-00703, observed at the light microscopic level byThioflavin T fluorometry and Congo red staining assays, was confirmed bynegative stain electron microscopy. A number of different experimentswere initialized to further test the efficacy of PTI-777 and PTI-00703on inhibition of Aβ 1-40 fibril formation. As an example in one study,50 μM Aβ 1-40 was incubated at 37° C. for 7 days in the absence orpresence of PTI-777 at an Aβ:compound weight ratio of 1:1, with aliquotstaken at 0, 1, 3 and 7 days of incubation for analysis by negative stainelectron microscopy. As shown in FIG. 7, amyloid fibrils were observedat 3 days of incubation of Aβ 1-40 alone (FIG. 7A, arrowheads). PTI-777(FIG. 7B), and to a lesser extent, PTI-00703 (not shown), were botheffective in preventing Aβ 1-40 amyloid fibril formation. Only amorphousnon-fibrillar material was observed when Aβ 1-40 was incubated in thepresence of PTI-777 (FIG. 7B, arrowheads). Similar observations on theability of PTI-777 to cause a disruption/disassembly of pre-formedAlzheimer's Aβ 1-40 and 1-42 fibrils were also verified using electronmicroscopy (not shown).

Example 10 Marked Inhibition of Aβ 1-42 Amyloid Deposition by PTI-777 ina Rodent Model of Aβ Fibrillogenesis

[0226] In one study, the Aβ amyloid inhibitory ability of PTI-00703 wasdirectly compared to PTI-777 (on a weight to weight basis) in a rodentmodel of Aβ 1-42 amyloid deposition. 25 μg Aβ 1-42, 25 μg Aβ1-42+PTI-777 or 25 μg Aβ 1-42+PTI-00703 were directly infused (usingAlzet osmotic pumps) into hippocampus for 1 week in adult Sprague-Dawleyrats (250-300 grams; 3 months old; n=9 per group). The Aβ:PTI-777 orAβ:PTI-00703 weight ratio was 1:5. Amyloid deposition was assessed bythe blind scoring of Congo red stained brain sections throughout theinfusion site using an arbitrary scale (from 0 to 5), as previouslydescribed (Snow et al, Neuron 12:219-234, 1994). The resultsdemonstrated that PTI-777 was a much more potent inhibitor of Aβ 1-42fibril deposition than PTI-00703 (FIG. 8). Whereas animals infused withPTI-00703 showed a 62.7% inhibition, animals infused with PTI-777demonstrated an impressive (p<0.005) 89.2% inhibition, of Aβ 1-42 fibrildeposition. This study indicated that PTI-777 was ˜26% more effectivethan PTI-00703 in inhibition of Aβ 1-42 fibril deposition in braintissue.

Example 11 Marked Inhibition of Astrocytosis by PTI-777

[0227] In all animal model studies as described above, tissue sectionsthroughout the infusion site were immunostained using a number ofdifferent glial marker antibodies to assess the effects of PTI-777 onboth microgliosis (i.e. anti-OX 42 or anti-MAC1) and astrocytosis (i.e.anti-GFAP). Brain tissues obtained from animals infused with Aβ 1-42only, or Aβ 1-42 +PTI-777, and immunostained with microglial markersdemonstrated positive infiltrating microglia at the infusion site (notshown), with no apparent reduction in microgilal infiltration byPTI-777. In fact, animals infused with PTI-777 actually demonstrated agreater number of infiltrating macrophages/microglia at the amyloidinfusion sites. This is expected since Uncaria tomentosa (i.e. Cat'sclaw), from which PTI-777 is derived, is a known inducer of macrophageactivation and infiltration (Keplinger et al, U.S. Pat. Nos. 4,844,901;4,940,725; Laus and Keplinger, J. Chromatography 662:243-249, 1994; Lauset al, Phytochem. 45:855-860, 1997).

[0228] As expected, animals infused with Aβ 1-42, or due to theplacement of the cannulae in hippocampus alone, showed a markedastrocytosis at the amyloid deposition and cannulae injury sites (FIGS.9A, B). Surprisingly, however, hippocampi from animals infused withPTI-777 showed a marked reduction in GFAP immunostaining (FIGS. 9C, D),suggestive of a marked inhibition of astrocytosis by PTI-777.

Example 12 Final Purification of the PTI-777 Active Components:Fractions F and J

[0229] The preparative HPLC work on PTI-777 described above resulted in11 major water-soluble active fractions, each of which contained atleast one major compound. Compositional and structural studies demandthat the major individual compounds within each fraction be purified tohomogeneity in sufficient quantity for NMR and other spectroscopicstudies (about 30-50 mg). Although each of the 11 fractions of PTI-777showed significant activity in the in vitro assays, fractions F, J and Hwere singled out for initial final purification based on their startingpurity as determined by analytical HPLC and the amount of material.These fractions were passed through the preparative HPLC column untilthey were deemed pure by comparison of the UTV diode array spectra takenat four points across the single chromatographic peak, and assessment.

Example 13 Fraction F Structurally Identified as Chlorogenic Acid

[0230] Fraction F was the first material to be purified in a quantitysufficient for structural elucidation work. Mass spectroscopy and NMRspectroscopic analysis were initially employed. Several different typesof mass spectra [(chemical ionization (CI), fast atom bombardment (FAβ),and electron impact (EI)] were taken of the purified sample. A clearspectrum that revealed the molecular weight of the compound was neverobtained under standard sampling conditions.

[0231] The ¹H-NMR (500 MHz) of fraction F in pyridine (d₅) showed 12signals (FIG. 10A). A broad signal at about 8.4 ppm was attributed to OHgroups on the compound. Two strongly coupled signals at 8.0 and 6.8 ppmand three aromatic signals at 7.5, 7.16 and 7.07 ppm were present in thespectrum, all indicative of H bound to sp² hybridized carbon atoms.There were also signals at 6.2, 4.75, 4.3, 2.9, 2.72 and 2.7 ppm. All ofthese signals with the exception of the ones at 8.4 and 2.72 ppm showedintegration consistent with one proton. The integration of the 2.72 ppmsignal was closer to 2 protons and the OH signal at 8.4 ppm was notintegrated. The Correlation Spectroscopy (COSY) spectrum (a twodimensional NMR experiment identifying adjacent protons) showed that theprotons responsible for the signals at 8.0 and 6.8 ppm were adjacent toeach other. The large coupling constant of these two signals as well astheir chemical shifts were indicative that these protons were attachedto a carbon-carbon double bond system with trans geometry coupled to anaromatic ring. The COSY spectrum also revealed that the three aromaticprotons were all on the same benzene ring and that the remaining sixsignals all showed connectivity indicating five contiguous carbon atoms.

[0232] The ¹³C NMR (500 MHz) in pyridine (d₅) showed 16 discrete signalregions (FIG. 10B). The two signals at 177 and 167 ppm indicated thepresence of two carbonyl C atoms. There were 8 carbons found in theshift range for sp² carbons (signals at 150, 147, 146, 127, 122, 117,116 and 115 ppm). Three of these carbons showed doubled signals (146,116 and 115 ppm). There were four signals (76, 74, 72 and 71 ppm)indicative of sp³ carbons bonded to oxygen atoms. Three of these signalswere doubled (74, 72 and 71 ppm). The two signals at 39.7 and 39.2 ppmwere representative of sp³ carbon atoms not bonded to oxygen. The sixdoubled signals were assumed to be due to a mixture of isomers. TheDistortionless Enhancement by Polarization Transfer (DEPT) experiment(distinguishes between carbon atoms bonded to 1, 2, 3 or no H atoms)indicated that the aromatic carbon atoms showing signals at 150, 147 and127 ppm were not bonded to H. This was also the case for the atomresponsible for the signal at 76 ppm. The Heteronuclear CorrelationSpectroscopy (HECTOR) experiment helped confirm which ¹H signals wereassociated with individual ¹³C signals.

[0233] The diode array ultraviolet (UV) spectrum of fraction F showed apeak at 330 nm indicating a possible presence of an aromatic ring withextended conjugation. Analysis of both the ¹H and ¹³C NMR data pointedto the presence of a tri-substituted aromatic ring with two phenolicgroups and a conjugated ethylene group with trans geometry. The chemicalshift of the carbonyl carbon at 177 pp, suggested that this signal wasdue to a carboxylic acid group attached to this conjugated system. TheCOSY data suggested a chain of five contiguous carbon atoms, three ofwhich were oxygenated. Attachment of the remaining sp³ quaternary carbonatom (76 ppm) to both ends of the five-carbon atom chain would form acyclohexane ring, which would be consistent with the data. The remainingcarbonyl carbon atom was assigned as a carboxylic acid group attached tothe cyclohexane ring. The conjugated aromatic and the cyclohexaneportions of the molecule were connected via an ester linkage, againconsistent with the chemical shift data.

[0234] The structural features of fraction F were consistent with thecompound, chlorogenic acid (C₁₆H₁₈O₉ MW 354.31)(FIG. 11). The structuralassignment of fraction F as chlorogenic acid was confirmed by comparisonof both the ¹H and ¹³C NMR spectra of Fraction F with the publishedspectra of chlorogenic acid [(“Aldrich Library of ¹³C and ¹H FT NMRSpectra” I (2) 1235C)]. The spectra were identical when solventdependent chemical shift changes were taken into account. The publishedUV spectrum of chlorogenic acid was also compared with that of fractionF and found identical. This study indicated that the major compoundwithin fraction F was identified as chlorogenic acid.

[0235] Chlorogenic acid can be found in green coffee beans, and alsooccurs with its isomers (isochlorogenic acid and neochlorogenic acid) infruit, leaves, and other tissues of dicotyledeneous plants. Thiscompound forms caffeic acid on hydrolysis (Barnes et al, J. Am. Chem.Soc. 72:4178-4182, 1950; Corse et al, Tetrahedron 18:1207-1210, 1962),and due to its polyphenolic content (see FIG. 12) turns black withferric chloride (Barnes et al, J. Am. Chem. Soc. 72:4178-4182, 1950;Harborne in Phytochemical Methods. A Guide to Modern Technicues andAnalysis., Chapman and Hill, London, N.Y., 1984, pp.38). Initial invitro testing of commercially available chlorogenic acid (Sigma/Aldrich)indicated that it is a potent Aβ fibrillogenesis inhibitor (not shown)as determined by Thioflavin T fluorometry and Congo red staining assaysas described herein.

Example 14 Successful Labeling of PTI-777 with 3H

[0236] In order to determine whether PTI-777 and/or its components havethe capacity to cross the blood-brain-barrier and enter the brain,PTI-777 had to be effectively radiolabeled without affecting itsstructure. Due to its highly electron rich structure due the presence ofOH groups such as those found in chlorogenic acid (i.e. fraction F),PTI-777 was initially labeled with tritium using proprietary atombombardment technologies. 0.9 mCi of ³H-PTI-777 with specific activityof 1.25 mCi/mg (at a concentration of 1 mCi/ml) was prepared and usedfor animal work. FIG. 12 demonstrates the HPLC profile of unlabelledPTI-777 (upper panel) which is nearly identical to the HPLC profile of³H-PTI-777, indicating that radiolabeling with tritium did little toalter the structure of PTI-777. The bottom panel of FIG. 12 demonstratesthe distribution of radioactivity measured in 0.5 ml fractions that wereeluted from the HPLC column and collected beginning at 16.5 minutes.This graph demonstrates that the HPLC peaks of PTI-777 also demonstratethe greatest radioactivity.

Example 15 Penetration of ³H-PTI-777 into Brain Tissue Following aSingle Intravenous Administration

[0237] In order to determine the potential ability of ³H-PTI-777 toenter into brain tissue (and cross the blood-brain-barrier), male andfemale Sprague-Dawley rats were administered with a single intravenousinjection of ³H-PTI-777. Two animals per group were then sacrificed at 5minutes, 1 hour, 6 hours and 24 hours following administration and theamount of ³H-PTI-777 in brain tissue was determined. As shown in FIG.13, ˜18,000 dpm/gram of brain tissue was found within 5 minutesfollowing intravenous administration. By 1 hour, this had decreased to˜10,100 dpm/g brain tissue. However, at 24 hours, the brain tissue stillcontained ˜7,000 dpm/gram brain tissue, indicating that ˜41% of theinitial dose found to enter the brain was retained in the brain tissueover a 24-hour period. This study suggested that the water-solubleamyloid inhibitory components of Uncaria tomentosa (i.e. PTI-777) havethe capability to enter the brain, an d thus are anticipated to beeffective for the inhibition of amyloid protein fibrillogenesis forcentral nervous system disorders, such as Alzheimer's disease, Down'ssyndrome, Parkinson's disease (with fibrils formed consisting ofalpha-synuclein), and other amyloid disease as described herein.

Example 16 Fraction J Structurally Identified as Epicatechin

[0238] Fraction J was the second material to be purified in a quantitysufficient for structural elucidation work. Following the isolation andpurification of PTI-777-compound “J”, spectroscopic studies on thecompound and its pentaacetate derivative revealed its structure to bethe same as epicatechin (C₁₅H₁₄O₆; FW 290.27). The results of thesespectroscopic studies are discussed below.

Mass Spectroscopy

[0239] PTI-777-Comoound J: Numerous attempts to obtain a reliablemolecular ion peak of the compound using both the fast atom bombardment(FAB+) and chemical ionization (CI) techniques were unsuccessful. Areliable molecular ion peak (M+1 with m/z of 291.05) was however,obtained using electrospay techniques using both time-of-flight (FIG.14) and fourier transform (FIG. 15) mass spectroscopy. This mass tocharge (m/z) ratio of 290 is consistent with a possible molecularformula of C₁₄H₁₂O₇ or C₁₅H₁₄O₆. An electron impact (EI) initiated massspectrum showed large m/z fragments at 123 (C₇H₇O₂), 139 (C₇H₇O₃), and152 (C₉H₈O₃)(FIG. 16).

[0240] Pentaacetate derivative of compound J: Mass spectra taken in boththe FAB+ (FIG. 17) and electron impact (EI) modes (FIG. 18) gavemolecular ion peaks of 523 and 500 respectively correlating with asodiated and non-sodiated pentaacetate derivative of a compound withmolecular weight 290. High resolution spectra taken in these modes gaveobserved m/z ratios of 523.1214 (C₂₅H₂₄O₁₁Na error −0.4 ppm/−0.2 mmu)and 500.1317 (C₂₅H₂₄O₁₁, error−0.2 ppm/−0.1 mmu). This informationfirmly established the molecular formula of the pentaacetate derivativeof PTI-777-compound J as C₂₅H₂₄O₁₁ and hence, compound Jto have themolecular formula corresponding to C₁₅H₁₄O₆.

Nuclear Magnetic Resonance (MNR) Soectroscopy

[0241] PTI-777-Compound J: In d₆ acetone, the ¹H NMR spectrum (FIG. 19)of PTI-777-compound J showed a doublet of doublets centered on 2.8 ppm(2H), a sharp multiplet at 4.2 ppm (1H), an apparent singlet at 4.85 ppm(1H), two singlets at 5.9 (1H) and 6.0 ppm (1H), two multiplets at 6.8ppm (1H), a sharp multiplet at 7.02 ppm (1H) and four hydroxy signals at7.83 (2H), 8.03 (1H), 8.2 (1H), and 3.6 ppm (1H). The ¹³C NMR spectrum(FIG. 20) displayed 15 signals at 29.06, 66.98, 79.49, 95.72, 96.20,99.85, 115.35, 115.53, 119.42, 132.35, 145.35, 145.46, 157.23, 157.65,and 157.65 ppm.

[0242] Both the ¹H (FIG. 21) and ¹³C (FIG. 22) spectra in D₂O with 0.1%triflouroacetic acid were significantly different form the abovereported spectra. The ¹H spectrum (FIG. 21) differed most strikingly inthe absence of the two singlets at 5.9 and 6.0 ppm. In the ¹³C spectrum(FIG. 22), the three signals between 90 and 100 ppm moved to just above160 ppm. The sample remained stable in the acidified D₂O when storedover the course of several months at room temperature, but it begins todegrade in acetone within 24 hours.

[0243] Correlation spectroscopy (COSEY) spectra of the sample in both d₆acetone (FIG. 23) and acidified D₂O (FIGS. 24A-C) revealed couplingbetween the 3 signals from 6.8 to 7.1 ppm and the signals at 2.8 and 4.2ppm.

[0244] Pentaacetate derivative of PTI-777-compound J: The expectedmethyl groups and carbonyl carbon atoms were present in both the protonand carbon spectra of the acetylated derivative of J. The most strikingchanges in the ¹H MNR spectrum (CDCl₃) upon acetylation (FIG. 25) werethe downfield shifts of about 0.6 ppm of the two singlets at 5.9 and 6.0ppm and the downfield shift of 1.2 ppm of the apparent singlet at 4.85ppm (compare to FIG. 21). The ¹³C spectrum (FIG. 26) was less affected,but the aromatic carbon atoms were, in general, shifted downfield uponacetylation. All 15 carbon signals associated with compound J werereadily seen in the spectrum.

[0245] In addition to the ¹H-¹H coupling detected in the COSEY spectrumof the unacetylated compound (FIG. 23, 24A-C), the derivative (FIG. 27)showed a connection between the apparent singlet at 4.85 ppm and thesharp multiplet at 4.2 ppm of the original compound. The heteronuclearcorrelation spectroscopy (HETCOR) spectrum (FIGS. 28A-28C) of theacetylated derivative was consistent with expectation and confirmed andthe assignment and the identity of the protonated carbon atoms.

[0246] Ultra Violet (UV) Spectroscopy: The UV spectrum ofPTI-777-compound J showed a maximum at 278 nm consistent with anaromatic phenolic compound (FIG. 29). Structural Assignment: The ¹³C NMRspectrum showed the presence of three sp³ type carbon atoms, 9 sp²carbon atoms (no carbonyl carbons) and 3 carbon atoms that were eithervery upfield sp² type or very downfield sp³ type carbon atoms. The ¹HMNR spectrum indicated the presence of 4 phenolic hydroxyl groups andone non-aromatic hydroxyl group. The major fragments observed in the EImass spectrum corresponded to dihyroxylated benzene rings without andwith carbon group substitution (m/z of 123, 139 and 152). The proton NMRdata (6.8-7.02 ppm) showed evidence for a tri-substituted benzene ringthat was hydroxylated. Comparison of ¹H NMR spectra data of modelcompounds with that of compound J, showed that the observed splittingpattern and chemical shifts of the signals were consistent with a1-substitued, 3,4-dihydroxy benzene structure.

[0247] The COSEY spectra showed ¹H-¹H coupling in a contiguousthree-carbon fragment. The relative chemical shifts of two of thesecarbon atoms indicated that they were directly bonded to oxygen atoms.The large chemical shift change observed for the signal at 4.85 ppm uponacetylation indicated the location of the non-aromatic hydroxyl groupand the chemical shift of the third carbon atom implied that it wasbenzylic. Together, these data were consistent with the followingstructure, Ar—CH₂—CH(OH)—CH(R)O.

[0248] The above fragments accounted for all but 4 hydrogen atoms (twoof which are found in phenolic type OH groups) and for all but 6 carbonatoms (3 sp² type and 3 intermediate between sp³ and sp² type). The C₆H₄formula indicated a high degree of unsaturation consistent with a secondaromatic ring. The remaining unaccounted for singlets in the ¹H NMR at5.9 and 6.0 ppm revealed that this ring was tetra substituted andelectron rich. These data indicated that this benzene ring was bonded tothree oxygen atoms (two hydroxy groups and one ether) and one carbonatom.

[0249] These three structural units (two phenolic rings and the threecarbon fragment) when connected together form a flavanol structureidentical to the diastereomers, catechin and epicatechin. Comparison ofthe ¹H and ¹³C NMR spectra, as well as the infrared (IR) spectra, ofcompound J with the published spectra (Aldrich collection) for catechinand epicatechin (FIGS. 31, 32) established the identity ofPTI-777-compound J as epicatechin. The splitting pattern of doublet ofdoublets center on 2.8 ppm in the ¹H NMR spectrum matches that of theepicatechin reference spectrum. The pattern of the aromatic signalsbetween 6.8 and 7.02 ppm is also most similar to that of epicatechin(allowance must be made for differences in solvents and spectrometerfield strengths). The IR spectrum of compound J matches closely withthat of epicatchin, while the IR spectrum of catechin differssignificantly in the fingerprint region.

Example 17 Compound “H” Demonstrates Potent Disassembly/Dissolution ofAβ 1-42 Fibrils

[0250] Further purification of fraction H led to the isolation of themajor compound within this fraction (known as compound H), which ran asa single peak on HPLC (not shown). The following methodology was used toisolate compound H from PTI-777. This methodology is different in someways to the previous examples presented in the specification.

[0251] PTI-777 was placed on an LH-20 column. The material was elutedwith successive column volumes of water/methanol mixtures containing0.1% TFA, beginning with 25% methanol and increasing to 100% menthol in25% increments. These fractions were analyzed by analytical HPLC, thosefractions containing the peak designated as “compound H”, were combined,and the solvent removed with the aid of a rotary evaporator. A DynamaxC-18, 5 m column (with dimensions of 4.6 mm×25 cm) was used foranalytical HPLC using a gradient of distilled water (solvent A) andmethanol (solvent B) each containing 0.1% TFA, with detection at 280 nm.The gradient conditions were 0.0 to 9.0 min (25% to 36% B gradient), 3.0to 10.0 min (36 to 100% B gradient), 10.0 to 12.0 min (100% B) and 12.0to 13.0 min (100 to 25% B gradient), all at a flow rate of 20.0 ml/min.

[0252] The resulting material was dissolved in water/methanol (80/20)containing 0.1% TFA and applied (150 mg/run) to a preparative HPLCcolumn (Dynamx C-18, 5 m, 21.4 mm×25 cm) fitted with an appropriateguard column. The following gradient conditions were used where solventA was water with 0.1% TFA and solvent B was methanol with 0.1% TFA. Thepreparative HPLC gradient conditions were 0.0 to 3.0 min (20% B to 25%B), 3.0 to 9.0 min (25 to 45% B), 9.0 to 10.0 min (45 to 100% B), 10.0to 12.0 min (100% B) and 12.0 to 13.0 min (100 to 25%), all at a flowrate of 20.0 ml/min. Detection was at 280 and 300 nm.

[0253] The H containing fractions obtained from the preparativecolumn,were reanalyzed by analytical HPLC and appropriately combined.They were then concentrated on a rotary evaporator and the remainingsolvent was removed using a Speed-Vac ® drying apparatus. The materialwas re-chromatographed using the preparative procedure outlined aboveuntil a pure material, known as “compound H”, was obtained as determinedby analytical HPLC and a dry sample of the compound was obtained asdescribed.

[0254] Initial characterization studies of “compound H” indicates thatit also contains an aromatic ring to which hydroxyl groups are attached(not shown)

[0255] Thus, “compound H” appears to belong to the group of compoundsknown as “polyphenolics”. Compound “H” was tested in direct comparisonto PTI-777 in order to determine its relative efficacy in causingdisassembly/disruption of pre-formed Aβ 1-42 fibrils. Although the exactstructure of this compound is unknown at this time, the data describedbelow indicates that “compound H” is potent in causingdisassembly/dissolution of pre-formed Aβ 1-42 fibrils and is thereforean important amyloid inhibiting component of PTI-777.

[0256] In one study, Thioflavin T fluorometry was used to determine theeffects of PTI-777, “compound H” and EDTA (as a negative control) ondisassembly/dissolution of pre-formed Aβ 1-42 fibrils. In this study, 25μM of pre-fibrillized Aβ 1-42 (Bachem Inc) was incubated at 37° C. for 1week either alone, or in the presence of PTI-777, “compound H”, or EDTAat an Aβ:test compound weight ratio of 1:1 (approximate molar ratio of1:10). Following a 1 week incubation 50 μl of each incubation mixturewas transferred into a 96-well microtiter plate containing 150 μl ofdistilled water and 50 μl of a Thioflavin T solution (i.e. 500 mMThioflavin T in 250 mM phosphate buffer, pH 6.8). The fluorescence wasread at 485 nm (444 nm excitation wavelength) using an Elisa platefluorometer after subtraction of buffer alone as blank. As shown in FIG.33, whereas EDTA caused no significant inhibition of Aβ 1-42 fibrils,PTI-777 demonstrated a significant 93.7% disruption/disassemblyofpre-formed Aβ 1-42 fibrils, whereas “compound H” caused a significant93.2% disruption/disassembly of pre-formed Aβ 1-42 fibrils, as indicatedby the marked lowering of fluorescence.

[0257] The disruption of Aβ 1-42, even in its monomeric form, wasconfirmed by a study involving the use of SDS-PAGE and Western blottingmethods. In this latter study, triplicate samples of pre-fibrillized Aβ1-42 (25 μM) was incubated at 37° C. for 1 week either alone or in thepresence of PTI-777 (1:1 wt/wt ratio), compound “H” (1:1 wt/wt ratio) orEDTA (1:1 wt/wt ratio). 5 μg of each sample was then filtered through0.22 μfilter. 1 μg of protein recovered from the filtrate was thenloaded per lane, and ran on 10-20% Tris-Tricine SDS-PAGE, blotted tonitrocellulose and detected by ECL using an anti-Aβ antibody (clone6E10; Senetek). As shown in FIG. 34, Aβ 1-42 was detected as a ˜4kilodalton band (i.e. monomeric Aβ) following incubation alone, or inthe presence of EDTA. Aβ 1-42 monomers were not detected following theincubation of Aβ 1-42 with either PTI-777 or “compound H” (FIG. 34)suggesting that both were capable of causing a disappearance ofmonomeric Aβ 1-42.

Further Aspects and Utilizations of the Invention TherapeuticApplications

[0258] One embodiment of the present invention is to formulate prior toadministration in a patient, a pharmaceutical formulation comprisingPTI-777 (and/or one or more of its individual components such aschlorogenic acid, epicatechin or “compound H”) in one or morepharmaceutical acceptable carriers, diluents or excipients. In apreferred embodiment, a patient who has Alzheimer's disease, type IIdiabetes, Parkinson's disease, or any other amyloidosis, would orallyconsume PTI-777 or components thereof in pill, tablet, caplet, soft andhard gelatin capsule, lozenge, vegicap, liquid drop, solution, syrup,tea bag, and/or bark powder form.

[0259] In another preferred embodiment PTI-777 (and/or one or more ofits individual components) in any form could be further modulated usingsuitable carriers, excipients and diluents including lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water syrup, methylcellulose, methyl and propylhydroxybenzoates, talc, magnesium stearateand mineral oil. The formulations can additionally include lubricatingagents, wetting agents, emulsifying and suspending agents, preservingagents, sweeting agents or flavoring agents. The compositions of theinvention may be formulated so as to provide quick, sustained or delayedresponse of the active ingredient after administration to the patient.The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 1 to about 1000 mg of PTI-777 (and/or oneor more of its individual components) more usually about 400 to about750 mg of PTI-777 (and/or one or more of its individual components).However, it will be understood that the therapeutic dosage administeredwill be determined by the physician in the light of the relevantcircumstances including the clinical condition to be treated, the organor tissues affected or suspected to be affected with amyloidaccumulation, and the chosen route of administration. Therefore, theabove dosage ranges are not intended to limit the scope of the inventionin any way. The term “unit dosage form” refers to physically discreteunits suitable as unitary dosages for human subjects and other mammals,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect, in associationwith a suitable pharmaceutical carrier.

[0260] The following formulation examples are illustrative only and arenot intended to limit the scope of the invention in any way. For eachformulation provided as an example, lowering or raising of the PTI-777(and/or one or more of its individual components) concentration willcause a proportional lowering or raising of the other ingredients asindicated. Hard gelatin capsules may be prepared by using 500 mg ofPTI-777 (and/or one or more of its individual components), 400 mg ofstarch, and 20 mg of magnesium stearate. The above ingredients are mixedand filled into hard gelatin capsules in 920 mg quantities.

[0261] A tablet is prepared by using 500 mg of PTI-777 (and/or one ormore of its individual components), 800 mg of microcrystallinecellulose, 20 mg of fumed silicon dioxide and 10 mg of stearic acid. Thecomponents are blended and compressed to form tablets each weighing 1230mg.

[0262] An aerosol solution is prepared by using 0.25 active ingredient,29.75 ethanol, and 70 of propellent 22 (chlorodifluoromethane). ThePTI-777 (and/or one or more of its individual components) is mixed withethanol. The mixture is added to a portion of the Propellent 22, cooledto −30oC., and transferred to a filling device. The required amount isthen fed to a stainless steel container and diluted with the remainderof the propellent. The value units (listed above) are then fitted to thecontainer. Such an aerosol form of PTI-777 (and/or one or more of itsindividual components) may be useful for the treatment of amyloidsinvolving the brain (such as Alzheimer's disease, Down's syndrome, priondiseases, Parkinson's disease etc) by using an aerosol or nasal spray.Previous studies have suggested that in these central nervous systemamyloidoses the initial form of entry of a possible environmental agentwhich may be playing a role in pathogenesis may be derived from theoutside world through the nasal passages.

[0263] Tablets are made by using 120 mg of PTI-777 (and/or one or moreof its individual components), 90 mg of starch, 70 mg ofmicrocrystalline cellulose, 8 mg of polyvinylpyrrolidone (as 10% inwater), 9 mg of sodium carboxymethyl starch, 1 mg of magnesium stearateand 1 mg of talc (total=300 mg). PTI-777 (and/or one or more of itsindividual components), starch and cellulose are passed through a No.45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50oC. and passed through a No. 18 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate and talc, previously passedthrough a No. 60 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 300 mg.

[0264] Capsules each containing 160 mg of medicant are made by using 160mg of PTI-777 (and/or one or more of its individual components), 118 mgof starch, 118 mg of microcrystalline cellulose, and 4 mg of magnesiumstearate (total=400 mg). The PTI-777 (and/or one or more of itsindividual components), cellulose, starch and magnesium stearate areblended, passed through a No. 45 mesh U.S. sieve, and filled into hardgelatin capsules in 400 mg quantities.

[0265] Suppositories each containing 225 mg of PTI-777 (and/or one ormore of its individual components) are made by using 225 mg of PTI-777(and/or one or more of its individual components), 2,000 mg of saturatedfatty acid glycerides (total=2,225 mg). The PTI-777 (and/or one or moreof its individual components) are passed through a No. 60 mesh U.S.sieve and suspended in the saturated fatty acid glycerides previouslymelted using the minimum heat necessary. The mixture is then poured intoa suppository mold of nominal 2 g capacity and allowed to cool.

[0266] Suspensions each containing 50 mg of medicant per 5 ml dose aremade by using 50 mg of PTI-777 (and/or one or more of its individualcomponents), 50 mg of sodium carboxymethyl cellulose, 1.25 ml of syrup,0.10 ml of benzoic acid solution, flavor, color, and purified water tototal 5 ml. The medicant is passed though a No. 45 mesh U.S. sieve andmixed with the sodium carboxymethyl cellulose and syrup to form a smoothpaste. The benzoic acid solution, flavor and color are diluted with someof the water and added, with stirring. Sufficient water is then added toproduce the required volume.

[0267] An intravenous formulation is prepared by using 250 mg of PTI-777(and/or one or more of its individual components), and 1000 mg ofisotonic saline. The solution of the above ingredients is administeredintravenously at a rate of 1 ml per minute to a subject in need oftreatment.

[0268] In a preferred embodiment the therapeutic compound of theinvention can be administered in any pharmaceutically acceptablevehicle. As used herein “pharmaceutically acceptable vehicle” includes,but is not limited to, any and all solvents, sterile liquids, such aswater and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like, dispersion media, coatings, antibacterial andantifungal agents, isotonic and adsorption delaying agents, and the likewhich are compatible with the activity of the compound and arephysiologically acceptable to the subject. An example of apharmaceutically acceptable vehicle is buffered normal saline (0.15molar NaCl). The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Supplementary activecompounds can also be incorporated into the fcompositions. Suitablepharmaceutical excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, fluor, chalk, silica gel, magnesium carbonate,magnesium stearate, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. These compositions can take the form of solutions,suspensions, tablets, pills, capsules, powders, sustained-releaseformulations and the like.

[0269] In the methods of the invention, amyloid formation, deposition,accumulation and/or persistence in a subject is inhibited byadministrating PTI-777 (and/or one or more of its individual components)in a therapeutic dosage to the subject. The term subject is intended toinclude living organisms in which amyloidosis can occur. Examples ofsubjects include humans, monkeys, cows, dogs, sheep, cats, mice, rats,and transgenic species thereof. Administration of the compositions ofthe present invention to a subject to be treated can be carried outusing known procedures, at dosages and for periods of time effective toinhibit amyloidosis in the subject. An effective amount of thetherapeutic compound necessary to achieve a therapeutic effect may varyaccording to factors such as the amount of amyloid already deposited atthe organ or tissue site in the subject, the age, sex and weight of thesubject, and the ability of the therapeutic compound to inhibit amyloidformation, deposition accumulation, persistence, and/or to causedissolution of pre-formed amyloid in the subject. Dosage regimens cantherefore be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the needs of the therapeuticsituation. A non-limiting example of an effective dose range for PTI-777(and/or one or more of its individual components) is between 400 and1000 mg/kg of body weight/per day.

[0270] Different modes of delivery of PTI-777 (and/or one or more of itsindividual components) may be used. Accordingly, a preferred route ofadministration is oral administration. Alternatively, PTI-777 (and/orone or more of its individual components) may be administered by othersuitable routes such as subcutaneous, intravenous, intraperitoneal, allroutes administered by injection. Depending on the route ofadministration, the active compound may be coated in a material toprotect the compound from the action of acids and other naturalconditions which may inactivate the compound.

[0271] To administer PTI-777 (and/or one or more of its individualcomponents), it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its activation.For example, the therapeutic compound may be administered to a subjectin an appropriate carrier, for example, liposomes or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes.

[0272] The PTI-777 (and/or one or more of its individual components) mayalso be administered parenterally or intraperitoneally. Dispersions canbe prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

[0273] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for thepreparation of sterile injectable solutions or dispersion. In all cases,the composition must be sterile and must be fluid to the extent thateasy use in the syringe exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thevehicle can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, prabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol,in the composition. Prolonged absorption of the injectable compositionscan be brought about by including in the composition an agent whichdelays absorption, for example, aluminum monostearate or gelatin.

[0274] Sterile injectable solutions can be prepared by incorporating thetherapeutic compound in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the therapeutic compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe therapeutic agent plus any desired ingredients from a previouslysterile-filtered solution thereof.

[0275] The PTI-777 (and/or one or more of its individual components) forAlzheimer's disease and other central nervous system amyloidoses may beoptimized to cross the blood-brain barrier, although initial studies asshown in Example 14 demonstrate that PTI-777 (and/or one or more of itsindividual components) has the ability to cross the blood-brain-barrierand enter the brain. However, if optimization is necessary, methods ofintroductions include but are not limited to systemic administration,parenteral administration i.e., via an intraperitoneal, intravenous,perioral, subcutaneous, intramuscular, intraarterial, intradermal,intramuscular, intranasal, epidural and oral routes. In a preferredembodiment, PTI-777 (and/or one or more of its individual components)may be directly administered to the cerebrospinal fluid byintraventricular injection. In a specific embodiment, it may bedesirable to administer PTI-777 (and/or one or more of its individualcomponents) locally to the area or tissue in need of treatment; this maybe achieved by, for example, and not by way of limitation, localinfusion during surgery, topical application, by injection, by infusionusing a cannulae with osmotic pump, by means of a catheter, by means ofa suppository, or by means of an implant.

[0276] In yet another embodiment PTI-777 (and/or one or more of itsindividual components) may be delivered in a controlled release system,such as an osmotic pump. In yet another embodiment, a controlled releasesystem can be placed in proximity to the therapeutic target, ie. thebrain, thus requiring only a fraction of the systemic dose.

INDUSTRIAL APPLICABILITY

[0277] Methods of isolation for the identification and purification ofthe potent amyloid inhibitory ingredients are disclosed. Use of suchextracts from the inner bark and root parts of Uncaria tomentosa andrelated plant materials are anticipated to be of enormous benefit tohuman patients with Alzheimer's disease, type II diabetes, Parkinson'sdisease and other amyloidoses throughout the world, due to thepreviously unknown ability of these compounds to inhibit amyloid fibrilformation, and cause disruption/ dissolution of pre-formed amyloidfibrils.

[0278] The disclosed methods are currently the only known methods bywhich to produce these previously unknown extracts and compounds. Theyare anticipated to be readily manufacturable in scaled up commercialproduction facilities, and made available to the world's population.

[0279] In compliance with the statute, the invention has been describedin language more or less specific as to structural features. It is to beunderstood, however, that the invention is not limited to the specificfeatures shown, since the means and construction shown comprisepreferred forms of putting the invention into effect. The invention is,therefore, claimed in any of its forms or modifications within thelegitimate and valid scope of the appended claims, appropriatelyinterpreted in accordance with the doctrine of equivalents.

We claim:
 1. A method for isolating compounds that possess amyloidinhibitory activity from plant matter of the genus Uncaria, the methodcomprising the steps: a) preparing a polar solvent extract of Uncariaplant matter, where the polar solvent extraction is selected from one ofthe extraction methods from the group of extraction methods consistingof extraction with water, extraction with a water solution of a polaralcohol, extraction with a water solution of acetonitrile and extractionwith a water solution of another polar organic solvent selected from thegroup of polar organic solvents consisting of triethanolamine, acetone,and the like, and running the extract through a first column thatcomprises hydroxy group containing resin, resin having hydrophobiccharacteristics but without any hydroxy groups, or a mixture of both; b)eluting the first column with distilled water, followed by eluting withnot more than 2-4 column bed volume washings with a dilute polaralcohol/water solution having an alcohol/water ratio not greater than50/50, and discarding any eluate; c) eluting the first column with oneor more column bed volume washings of a polar alcohol/water solutionhaving an alcohol/water ratio between 50/50 and substantially purealcohol, and collecting and drying the eluted volumes to a driedmaterial.
 2. The method of claim 1 wherein the column that compriseshydroxy containing resin, resin having hydrophobic characteristics butwithout any hydroxy groups, or a mixture of both is a column selectedfrom the group of columns consisting of C2 column, C4 column, C18column, and the like carbon-containing columns, Tris-acrylate column,LH-20 column, Affi-prep 10 gel column, and the like.
 3. The method ofclaim 1 wherein the polar alcohol/water solution has an alcohol/waterratio of 75/25 or higher.
 4. The method of claim 1 wherein the washingin step (c) is effected with substantially pure methanol.
 5. The methodof claim 1 wherein the plant matter of the genus Uncaria is taken fromone or more of the plants of the various Uncaria species plant groupconsisting of tomentosa, attenuata, elliptica, guianensis, pteropoda,bernaysli, ferra DC, kawakamii, rhyncophylla, calophylla, gambir, andorientalis.
 6. The method of claim 1 wherein the plant matter of thegenus Uncaria is taken from Uncaria tomentosa.
 7. The method of claim 6wherein the Uncaria tomentosa plant matter is taken from one or more ofthe group of plant parts consisting of inner bark and root.
 8. Themethod of claim 1 further comprising the steps: d) applying an aqueoussolution of the dried material from step (c) to a second columncomprising a hydrophobic resin, the second column having beenpreparatorily equilibrated in a solvent comprising about 95% water/5%acetonitrile, referred to herein as solvent A, and then eluting thesecond column with more solvent A and discarding the eluate; e) elutingthe second column with a mixture of solvent A containing 10-15% of asolvent comprising about 95% acetonitrile/5% water, referred to hereinas solvent B, and collecting and drying the eluted volumes to a driedmaterial.
 9. The method of claim 8 wherein the second column comprisinga hydrophobic resin is a column selected from the group of columnsconsisting of C18 SPE, Varian Chroma..Zone ™, other HPLC columns, othercarbon-containing columns, and the like.
 10. The method of claim 1 or 8further comprising the steps: f) making one or more injections of asolution of the dried material of step (c) or the dried material of step(e) in a solvent selected from the group of solvents consisting ofwater, water/dilute alcohol and solvent A comprising no more than 10%solvent B, into an HPLC instrument having a diode array uv/vis detectorwith a graphic display, the HPLC instrument further comprising areverse-phase column; g) eluting the material through the HPLC columnusing a solvent gradient profile as follows: 10% solvent B for about thefirst 20 minutes from start of elution, 10 to 100% solvent B gradientfor about minutes 20 to 30 from start of elution, and 100 to 10% solventB gradient for about minutes 30 to 32 from start of elution, whileobserving the uv/vis detector graphic display during the elutiongradient over time, and separating fractions of the eluate at elutiontimes corresponding to times associated with the graphic display peaks.11. The method of claim 10, wherein the reverse-phase column hasdimensions of about 2.2 cm×25 cm and contain about 95 ml of C18 reversephase resin, wherein the solution of the dried material is a solution ofabout 50 mg of the dried material of step (c) in about 1-2 ml of solventA, wherein the step of injecting the solution of dried material into theHPLC may be repeated, wherein a HPLC column solution gradient flow rateis set to about 5 mls per minute, and further wherein the solventgradient profile is 10% solvent B for 0 to 20 minutes, followed by 10 to100% solvent B gradient for minutes 20 to 30, and 100% to 10% solvent Bgradient from minutes 30 to 31; such that fractions F though N of theeluate are collected at the following times: fraction G (13-14 minutes),fraction F (15-16 minutes), fraction H (17-20 minutes), fraction I (21minutes), fraction J (22 -23 minutes), fraction K1 (24 minutes),fraction K2 (25 minutes), fraction L (26-27 minutes), fraction M (27-28minutes), and fraction N (28-29 minutes).
 12. The method of claim 10,wherein the reverse-phase column with dimensions of 1.0 cm×25.0 cmcontaining 20 ml of C18 reverse phase resin, wherein the solution of thedried material of step (c) is a solution of 50 μg of the dried materialin 50-100 μl of solvent A, wherein the step of injecting the solutioninto the HPLC is repeated multiple times, wherein a HPLC column solutiongradient flow rate is set to about 1.5 mls per minute, and furtherwherein the solvent gradient profile is 10% solvent B for 0 to 20minutes, followed by 10 to 100% solvent B gradient for minutes 20 to 30,and 100% to 10% solvent B gradient from minutes 30 to 31; such thatfractions F though O of the eluate are collected at the following times:fraction G (12-13 minutes), fraction F (13-14 minutes), fraction H (15minutes), fraction I (16 minutes), fraction J (18-19 minutes), fractionK1 (20 minutes), fraction K2 (21 minutes), fraction L (21-23 minutes),fraction M (23 minutes), fraction N (24 minutes), and fraction O (26-27minutes).
 13. The method of claim 10 wherein steps (f) and (g) are asfollows: f) injecting a solution of 1 gram of the dried material of step(c) in 5-10 ml of solvent A into an HPLC instrument having a Varianmodel 320 uv/vis detector set at 230 nm with a graphic display, the HPLCfurther comprising a 4.14 cm×25 cm Varian Dynamax column furthercomprising 380 ml of C-18 reverse phase resin, the column fitted to aVarian Prostar 215 solvent delivery system, or the like. g) eluting theHPLC column at a solution gradient flow rate of about 50 ml/minute, andfurther wherein the solvent gradient profile is with a solvent C/solventD gradient as follows: 0-4 minutes, 25% D; 4-11 minutes, 25-30% Dgradient; 11-14 minutes, 30-90% D gradient; 14-17 minutes, 90% D; and17-19 minutes, 90-25% D gradient, where C is water and D is methanol,such that fractions F through O of the eluate are separated at elutiontimes corresponding to times associated with the graphic display peaks.14. The method of claim 1 wherein the preparation in step (a)of theextract of Uncaria is as follows: 1) adding 4000 ml of methanol to 1 kgof Uncaria tomentosa and mixing 2) centrifuging the mixture at ×2,500 gusing a centrifuge for 30 minutes and collecting the supernatant; 3)extracting the insoluble material about 3 more times as steps a and babove; 4) combining the supernatants and evaporating to a dried extract,or to at least about 500 ml volume, using a rotary evaporator at 50° C.;5) washing the dried extract, or the 500 ml volume, 4 times with 300 mlof petroleum ether, and discarding the ether layer; 6) furtherevaporating any remaining methanol to dryness using a rotary evaporatorat 50° C.; 7) extracting the dried extract 5 times with 150 ml ofdistilled water, followed by centrifugation at 2,500×g for 30 minuteseach time, and 8) combining the supernatants and then lyophilizing usinga freeze-dryer.
 15. The method of claim 14 wherein the furtherpreparation of the extract of Uncaria from the resulting lyophilizedextract includes the following additional steps: 9) dissolving theresulting lyophilized extract into about 500 ml of distilled water, andapplying 50-100 ml portions to a 400 ml LH-20 column equilibrated withdistilled water. 10) eluting the LH-20 column with 1,100 ml of distilledwater (˜3 column volumes) and discarding the amber/yellow, non-activefractions; 11) eluting the LH-20 column with 1,100 ml of 100% methanol(˜3 column volumes) and collecting a set of active fractions andevaporating to dryness using a rotary evaporator at 50° C.
 16. Themethod of claim 8 wherein the aqueous solution of a dried material fromstep (c) is further prepared by the following steps: 1) dissolving thedried material in water at 80 mg/ml and applying 5 ml at a time to adisposable C18 SPE column (10 gram) equilibrated in a first solventcomprising about 95% water/5% acetonitrile/ 0.1% TFA; 2) washing with 3column bed volumes of the first solvent and discarding the eluate. 3)eluting with 3 column bed volumes of the first solvent furthercomprising about 12.5% of a second solvent comprising about 95%acetonitrile/5% water/0.1% TFA, and 4) lyophilizing the correspondingfractions using a freeze-dryer.
 17. The method of claim 8 wherein theaqueous solution of a dried material from step (c) is further preparedby the following steps: 1) dissolving the lyophilized fractions at 5grams in 20 ml water and applying 20 ml at a time to a VarianChroma..Zone ™apparatus 2) washing with 3 column bed volumes of a firstsolvent comprising about 95% water/5% acetonitrile/0.1% TFA anddiscarding the eluate; 3) eluting with 3 column bed volumes of the firstsolvent further comprising about 12.5% of a second solvent comprisingabout 95% acetonitrile/5% water/0.1% TFA, and 4) collecting and dryingthe next 3 column bed volumes of eluate.
 18. A method for isolatingwater-soluble components from Uncaria tomentosa that possess amyloidinhibitory activity, the method comprising the steps: a) adding 4000 mlof methanol to 1 kg of Uncaria tomentosa and mixing b) centrifuging themixture at ×2,500 g using a centrifuge for 30 minutes and collecting thesupernatant; c) extracting the insoluble material about 3 more times assteps a and b above; d) combining the supernatants and evaporating todryness (or until about 500 ml volume is reached) using a rotaryevaporator at 50° C., e) taking the powdered extract (or about 500 mlvolume), washing 4 times with 300 ml of petroleum ether, and discardingthe ether layer, f) evaporating the methanol to dryness using a rotaryevaporator at 50° C.; g) extracting the solid material 5 times with 150ml of distilled water, followed by centrifugation at 2,500×g for 30minutes each time; h) combining the supernatants and then lyophilizingusing a freeze-dryer; i) dissolving the resulting lyophilized extractinto about 500 ml of distilled water, and applying 50-100 ml portions toa 400 ml LH-20 column equilibrated with distilled water. j) eluting theLH-20 column with 1,100 ml of distilled water (˜3 column volumes) anddiscarding the amber/yellow, non-active fractions; k) eluting the LH-20column with 1,100 ml of 100% methanol (˜3 column volumes) and collectinga set of active fractions and evaporating to dryness using a rotaryevaporator at 50° C.; l) dissolving the fractions of step k in water (80mg/ml) and applying 5 ml at a time to a 10 gm disposable C18 SPE columnequilibrated in solvent A (solvent A is 95% water/5% acetonitrile/0.1%TFA); m) washing the column with 3 volumes of solvent A and discardingthe eluate; n) eluting the column with 3 volumes of solvent A containing12.5% solvent B (solvent B is 95% acetonitrile/5% water/0.1% TFA) andlyophilizing the eluate; o) taking 50 mg of the lyophilized eluate ofstep n and injecting multiple times into a Hewlett-Packard 1100 SeriesHPLC instrument with diode array detector, fitted with a 2.2 cm×25 cmVydac 218TP1022 C18 reverse-phase column maintained at 25° C. and at aflow rate of 5 ml/min; p) eluting the sample with the following solventprofile, 10% B for 0 to 20 minutes, 10-100% B gradient for minutes 20 to30, and 100-10% B gradient for minutes 30-31, where B is 95%acetonitrile/5% water/0.1% TFA; q) and separating and collecting thefractions into 11 major components defined as fraction G (13-14minutes), fraction F (15-16 minutes), fraction H (17-20 minutes),fraction I (21 minutes), fraction J (22-23 minutes), fraction K1 (24minutes), fraction K2 (25 minutes), fraction L (26-27 minutes), fractionM (27-28 minutes), and fraction N (28-29 minutes).
 19. A compositionfurther referred to herein as PTI-777 made according to the process ofclaims 1, 8, 10-13 or
 18. 20. A composition further referred to hereinas a PTI-777 fraction, the fraction selected from the group of fractionsconsisting of PTI-777 fraction G, PTI-777 fraction F, PTI-777 fractionH, PTI-777 fraction ., PTI-777 fraction J, PTI-777 fraction K₁, PTI-777fraction K₂, PTI-777 fraction L, PTI-777 fraction M, PTI-777 fraction N,and PTI-777 fraction O, wherein the selected fraction is made accordingto the process of any of the claims 1, 8, 10-13.
 21. The composition ofclaim 20 wherein the PTI-777 fraction selected from the group offractions is PTI-777 fraction H.
 22. The method of claim 1 furthercomprising the steps: d) applying an aqueous solution of the driedmaterial from step (c) to a second column, LH-20 or the like, elutingthe material from the column with successive column volumes ofwater/methanol mixtures containing 0.1% TFA, beginning with 25% methanoland increasing to 100% menthol in 25% increments, and collecting andcombining the fractions; e) separating, combining and drying a fractionto a dried material, referred to hereafter as compound H, by analyticalHPLC, the fraction containing a peak occurring between 7-8 minutes fromstart of elution on a Dynamax 5 μ C-18 column having dimensions of about4.6 mm×25 cm, using an elution gradient of water for solvent A andmethanol for solvent B, A and B each containing about 0.1% TFA, withdetection at 280 nm, the gradient conditions being 0 to 9 min fro 25% to36% B gradient, 3 to 10 min for 36 to 100% B gradient, 10 to 12 min for100% B and 12 to 13 min for 100 to 25% B gradient, all at a flow rate ofabout 20 ml/min; f) making one or more injections of a solution of thedried material of step (e) above in a solvent comprising water/methanol80/20 containing about 0.1% TFA and applied at about 150 mg/run to apreparative HPLC Dynamax 5 μ C-18 column with dimensions of about 21.4mm×25 cm, using substantially the same elution gradient as used in step(e) above, with detection at 280 and 300 nm, the gradient conditionsbeing 0 to 3 min for 20% to 25% B gradient, 3 to 9 min for 25 to 45% Bgradient, 9 to 10 min for 45 to100% B gradient, 10 to 12 min for 100% Band 12 to 13 min for 100 to 25%B gradient, all at a flow rate of about20 ml/min, the compound H fraction eluting between 7-8 minutes fromstart of elution, and; g) repeating steps (e) and (f) above until thepeak as seen on analytical HPLC in step (e) is relatively pure.
 23. Acomposition further referred to herein as compound H made according tothe process of claim
 22. 24. A method of treatment, prevention ormanagement of an amyloidosis, or a disease related to alpha-synuclein,in a mammalian subject susceptible to, or afflicted by, the amyloidosisor alpha-synuclein disease, the method comprising the step ofadministering to the subject a therapeutic amount of the composition ofclaim 12 and/or claim
 23. 25. A method of treatment, prevention ormanagement of an amyloidosis, or a disease related to alpha-synuclein,in a mammalian subject susceptible to, or afflicted by, the anyloidosisor alpha-synuclein disease, the method comprising the step ofadministering to the subject a therapeutic amount of fraction G,fraction F, fraction H, fraction I, fraction J, fraction K₁, fractionK₂, fraction L, fraction M, fraction N and/or fraction O compositions ofclaim
 20. 26. The method of claim 24 wherein the amyloidosis has anassociated amyloid and the amyloidosis is selected from the group ofamyloidoses associated with Alzheimer's disease, Down's syndrome,hereditary cerebral hemorrhage with amyloidosis of the Dutch type, theamyloidosis associated with type II diabetes, the amyloidosis associatedwith chronic inflammation, various forms of malignancy and FamilialMediterranean Fever, the amyloidosis associated with multiple myelomaand other B-cell dyscrasias, the amyloidosis associated with the priondiseases including Creutzfeldt-Jakob disease, Gerstmann-Strausslersyndrome, kuru and animal scrapie, the amyloidosis associated withlong-term hemodialysis and carpal tunnel syndrome, the amyloidosisassociated with endocrine tumors such as medullary carcinoma of thethyroid, and the alpha-synuclein associated diseases includingParkinson's disease and Lewy body disease.
 27. The method of claim 24wherein the amyloidosis is associated with Alzheimer's disease.
 28. Themethod of claim 26 wherein the associated amyloid is beta-amyloidprotein or Aβ, AA amyloid or inflammation-associated amyloid, ALamyloid, amylin or islet amyloid polypeptide, PrP amyloid,beta₂-microglobulin amyloid, transthyretin or prealbumin, or variants ofprocalcitonin.
 29. A method for the treatment, inhibition, prevention ormanagement of amyloid fibril or alpha-synuclein fibril formation,deposition, accumulation, aggregation and/or persistence in a mammaliansubject, the method comprising the step of administering to the subjecta therapeutic amount of the composition of claims 19, 20 or
 23. 30. Themethod of claim 29 wherein the route of administration of the method oftreatment is selected from the group consisting of oral administration,parenteral injection, intraperitoneal injection, intravenous injection,subcutaneous injection, or aerosol spray administration.
 31. Apharmaceutical agent comprising a therapeutically effective amount of amaterial made according to the process of claims 1, 8, 10-13, 18 or 22,the therapeutic amount of the material selected for efficacy in treatingan amyloid disease in a patient.
 32. A pharmaceutical agent comprising atherapeutically effective amount of a compound selected from the groupconsisting of chlorogenic acid and epicatechin, the compound and thetherapeutic amount of the compound selected for efficacy in treating anamyloid disease in a patient.
 33. The pharmaceutical agent of claim 31or 32 wherein the therapeutically effective amount of a materialcomprises a dosage in the range of from about 10 to 1,000 mg/kg of bodyweight of the patient.
 34. The pharmaceutical agent of claim 33 whereinthe therapeutically effective amount of a material comprises a dosage inthe range of from about 10 to 100 mg/kg of body weight of the patient.35. The pharmacological agent of claim 33 wherein said amyloid diseasefor treatment is selected from the group of amyloid diseases associatedwith Alzheimer's disease, Down's syndrome, hereditary cerebralhemorrhage with amyloidosis of the Dutch type, the amyloidosisassociated with type II diabetes, the amyloidosis associated withchronic inflammation, various forms of malignancy and FamilialMediterranean Fever, the amyloidosis associated with multiple myelomaand other B-cell dyscrasias, the amyloidosis associated with the priondiseases including Creutzfeldt-Jakob disease, Gerstmann-Strausslersyndrome, kuru and animal scrapie, the amyloidosis associated withlong-term hemodialysis and carpal tunnel syndrome, the amyloidosisassociated with endocrine tumors such as medullary carcinoma of thethyroid, and the alpha-synuclein associated diseases includingParkinson's disease and Lewy body disease.
 36. The pharmacological agentof claim 35 wherein said amyloid disease for treatment is Alzheimer'sdisease.
 37. The pharmacological agent of claim 33 further comprising apharmaceutically acceptable carrier, diluent, or excipient.
 38. Thepharmacological agent of claim 33 wherein the therapeutically effectiveamount of the material has an amyloid inhibitory activity or efficacygreater than 50%.